All 13 modern trials concluded that phage therapy was safe. Six of the 13 modern trials were exclusively safety trials. Seven modern trials investigated both safety and efficacy; efficacy was observed in two. Two of three historical trials did not comment on safety, while adverse effects in the third likely reflected the use of phage preparations contaminated with bacterial debris. None of the historical trials contained evidence of efficacy. The evidence from trials is that phage therapy is safe. For efficacy to be observed a therapeutic amount of the right phage(s) must be delivered to the right place to treat infections containing enough susceptible bacterial cells. Trials that have not demonstrated efficacy have not fulfilled one or more elements of this principle.Trials of phage therapy have not consistently reported efficacy. This contrasts with promising efficacy rates from a sizeable and compelling body of observational literature. This systematic review explores the reasons why many phage trials have not demonstrated efficacy. Four electronic databases were systematically searched for safety and/or efficacy trials of phage therapy. Sixteen trials of phage therapy were included, in which 378 patients received phage. These were divided into historical (pre-2000; N = 3;  Phages ns alone . We therThe use of carefully selected naturally occurring lytic phage(s) to treat bacterial infections is known as phage therapy. The first recorded instance of phage therapy occurred in Paris in 1919 . The widThere is a compelling body of observational evidence supporting the safety and efficacy of phage therapy ,11,12,13This systematic review will collate and critically appraise the results of all available clinical and safety trials of phage therapy. The objective of this review is not to derive summary statistics about the safety and efficacy of phage therapy. Instead, this review will explore the question of why trials may not have yielded efficacy data in line with compelling observational data. This analysis will inform a subsequent discussion of the challenges presented by clinical trials of phage therapy. ® Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Ovid MEDLINE® Daily, Ovid MEDLINE and Versions® (1946–2021) and Web of Science (1900–2021). The Web of Science Core Collection Citation Indexes searched were: Science Citation Index Expanded (1900–2021), Book Citation Index– Science (2005–2021) and the Emerging Sources Citation Index (2015–2021). The search was performed using the following terms: (phage OR bacteriophage) AND . In Ovid these terms were followed by the suffix ‘.mp.’ and they were searched as topics in Web of Science. A study protocol was not prepared or published prior to this study.Three electronic databases were searched for articles published up to and including 18 October 2021: EMBASE (1980–2021), Ovid MEDLINEThe raw search results were deduplicated using Endnote (version X8.0.1). The remaining studies underwent title and abstract screening. Eligible studies were explicitly clinical or safety trials of phage therapy and were published in the English language. Trial protocols and reports of pre-trial pilot studies were excluded. There were no limitations on study date or location. Studies identified as eligible by title and abstract screening were subsequently accessed in full to ensure they fulfilled the inclusion criteria. Studies which could not be accessed in full, including presentation abstracts, were excluded; their authors were not contacted. Title and abstract and full-text screening were performed independently by the authors (SDS or HJS and JDJ), with discrepancies resolved by agreement or, where necessary, a third author (SDS or HJS). This review was conducted in accordance with the PRISMA  guidelines , and a PThe following information was extracted from each eligible study into a spreadsheet: publication year; author(s); study location; study type; number of patients; pathogen(s); condition details; bacterial sensitivity to phage(s); details of the phage(s) used; trial treatment schedule and route(s), assays used to monitor safety and efficacy and details of other ongoing therapies  where reported; efficacy findings; comments or data regarding safety and adverse effects. All eligible studies were critically assessed using a Joanna Briggs Institute checklist for clinical trials . Data exn = 5), not published in English and/or not available in full (n = 2), a trial protocol (n = 1) or deemed to be compassionate use and not a clinical trial (n = 1). Patients in the latter report were treated with intravenous phage for severe staphylococcal infection. Nine patients were treated under an Australian ‘compassionate-use special access scheme’ [Systematic searching of three databases yielded 2485 articles, from which deduplication removed 583. The titles and abstracts of the remaining 1902 articles were screened against the selection criteria. Title and abstract screening identified 26 eligible articles, nine of which were excluded after full-text screening. Articles were excluded at the full-text stage because they were: not a trial of the safety or efficacy of phage therapy , the United States (n = 2), France and/or Belgium (n = 2), Australia (n = 1), Georgia (n = 1) and the UK (n = 1). Of the 13 trials, four were designated as ‘phase I’, two as ‘phase I/II’ and seven did not contain any explicit designation. The trials covered the administration of phage orally (n = 6), topically (n = 3), both orally and intra-nasally (n = 1), intra-aurally (n = 1), intra-nasally (n = 1) and by intra-vesicular washing (n = 1). No pre-phage gastric neutralisation was employed in any of the seven studies which administered oral phage. However, phage was suspended in weakly alkaline mineral water in five of the seven trials which used orally administered phage. The diverse routes of administration reflect the diverse range of pathogens and clinical conditions to which phages have been applied. Phages were applied as cocktails (n = 11), single-phage preparations (n = 1) or both cocktails and single-phage preparations (n = 1). Six of the 13 trials were safety trials among healthy adults and children. The remaining seven studies examined the efficacy of phage therapy for burn wound infections caused by P. aeruginosa and/or S. aureus (n = 2); diarrhoea caused by E. coli (n = 1); chronic otitis caused by P. aeruginosa (n = 1); staphylococcal chronic rhinosinusitis (n = 1); complex urinary tract infection caused by various pathogens (n = 1); or chronic venous leg ulcer patients in whom the presence of infection was not a criterion for inclusion (n = 1). Patients received, or were able to receive, simultaneous antibiotic therapy in three of the seven efficacy trials. Among the seven efficacy trials, prospective investigation of the bacterial sensitivity to phage therapy was undertaken in three trials, retrospective investigation in two and no investigations in two trials. Together, excluding control groups, the modern trials represented the application of phages to 302 patients from the ages of six months old, of which 156 were healthy/uninfected and 146 had a relevant bacterial infection.The 13 modern clinical or safety trials identified were published between 2005 and 2021 ,28,29,309 phages per gram of human faeces [Given the diverse array of phages, treatment regimens and routes of administration, the 13 modern clinical and safety trials included in this review provide a broad overview of the safety of phage therapy. Representing the administration of a range of phages in high concentrations by a variety of routes and, having used a comprehensive array of appropriate safety monitoring assays, all 13 of the modern trials concluded that phage therapy was safe and without phage-related adverse effects. This will be reassuring to readers less familiar with phage therapy. However, that trials have consistently demonstrated the safety of phage administration by a variety of routes is perhaps unsurprising when the evolutionary context is considered. Phages are ubiquitous in the environment and form a significant part of commensal human flora. For example, there are an estimated > 10n faeces . Phage gn faeces ,33. TogePseudomonas with one dose of a six-phage cocktail and observed significant clinical improvements from baseline and lower bacterial counts in the phage-treated group compared to the control group [S. aureus and in 2/9 bacterial eradication was achieved [Six of the 13 modern trials included in this review were safety trials and did not evaluate efficacy. Of the remaining seven trials, two demonstrated the efficacy of phage therapy in the context of chronic antibiotic-refractory infections ,29. In 2ol group . More reThe remaining five clinical trials were published over the last seven years and did not adequately demonstrate the efficacy of phage therapy. Examination of these trials shows that in hindsight these results are not a comment on the mechanistic efficacy of phages in killing bacteria, but instead reflect ongoing clinical and technical challenges facing phage therapy trials. In 2009, Rhoads and colleagues undertook a phase I safety trial of topical phage therapy among patients with chronic venous leg ulcers . The rep2 of phage cocktail was administered to half of a patient’s wound area. However, the patients experienced a delay of up to seven days in admission to the trial. In that time all the patients were receiving appropriate antibiotic therapy. Consequently, comparison of the bacterial loads between the phage treated and standard care halves of the wounds did not reveal any notable difference. Moreover, the authors noted that it was not expected that a single dose of topical phage therapy would elicit any conclusive proof of phage efficacy and rather the significance of the trial lay in its very undertaking. In 2014, Rose and colleagues undertook a small-scale clinical trial of topical phage therapy for burn-wound infection . A singlE. coli. No intestinal amplification of phages was observed, including in patients with phage-sensitive strains of E. coli, and the diarrhoea was attributed to streptococcal overgrowth in some patients. The authors considered that for those patients with sensitive E. coli there were likely too few E. coli organisms to sustain sufficient phage replication. Moreover, it is considered to be advantageous to administer gastric neutralisation prior to oral phage therapy [In 2016, Sarker and colleagues treated 78 children with diarrhoea, aged 6–24 months, with one of two oral phage cocktails . The tre therapy . However6 PFU/mL to a subtherapeutic 102 PFU/mL. This rendered PhagoBurn at best a safety trial as, although many phage particles were inactive, the equivalent of 106 PFU/mL of phage proteins were nonetheless administered topically. Aside from the phage concentration, it is notable that the infecting P. aeruginosa strains were of varying antibiotic sensitivity which could have complicated delineation of the relative antimicrobial effects of phage and antibiotics. Moreover, during the trial, it was found that three of the 10 participants in the phage group had P. aeruginosa colonies resistant to phage on day 0 of the trial. PhagoBurn therefore also illustrates the microbiological complexities associated with phage therapy and varying levels of resistance to antibiotics and phages that have the potential to complicate clinical trial results. In 2019, the much-anticipated results of the PhagoBurn trial were published, however the trial did not yield evidence of efficacy for several reasons . CrucialMost recently, in 2021, Leitner and colleagues published a clinical trial of intra-vesicular phage therapy for patients with complex urinary tract infection . DespiteIn summary, the four recent clinical trials that have failed to demonstrate the efficacy of phage therapy have done so because they have not achieved the ‘Goldilocks’ constellation of getting the right amount of the right phage(s) to the right place to treat infections containing enough susceptible bacterial cells. If just one of these factors is sub-optimal the efficacy of phage therapy will be dramatically reduced. However, the success of other clinical trials shows that it is possible to achieve a favourable constellation of these factors. However, such can be the variation between infections that achieving the appropriate constellation of these factors is arguably simpler on a case-by-case basis. This explains the notably greater success achieved by personalised clinical applications of phage therapy, in which both the phage(s) and clinical approach are tailored precisely to a patient’s individual clinical needs.Three historical trials of phage were identified by the systematic search ,37,38. TTwo of the three historical trials, published in 1966 and 1971, did not comment on safety or adverse effects ,37. The Historical users of phage were unable to purify their phage preparations. Impure phage preparations, such as raw staphylococcal phage lysate, contain a mixture of dead (lysed) bacterial cells, phage particles and bacterial growth media. Although a reaction to phage cannot be ruled out it is unlikely given the absence of adverse effects from modern clinical trials. Instead, the reactions reported most likely reflect a combination of an injection-site reaction and immune response to the substantial quantity of staphylococcal debris. Similar reactions have been observed in other 20th Century clinical reports of the use of subcutaneously injected raw phage lysates ,40. TherS. aureus. There is also insufficient information in the report to confirm the nature of the pathologies in question; for example, we now consider acne to be a multifactorial complex pathology, rather than a straightforward bacterial infection [None of the three historical trials reported evidence of efficacy. Bryant and colleagues evaluated the efficacy of intramuscular phage injections for the treatment of chronic furunculosis among children . It is nnfection . Third, S. aureus between the two groups reflects the lack of impact that phage administered by IM injection could reasonably be expected to have on nasopharyngeal colonisation, without considering the prior absence of bacterial culture and phage sensitivity analysis. Wittig and colleagues also evaluated the efficacy of injected staphylococcal phage lysate, in this case for the treatment of infective childhood asthma . The outV. cholerae. Although phage sensitivity testing was undertaken it was not clear whether it was pro- or retrospective. Nonetheless, the results of the phage sensitivity testing showed that while all classical strain isolates were susceptible to the phages used, the El Tor isolates exhibited varying degrees of substantial resistance. The authors noted that it is difficult to predict the phage sensitivity of Vibrio strains and suggested incompatibility between the phage and bacterial strain as an explanation for the El Tor results. Of greater importance however, and neatly accounting for the failure of phage therapy among the patients with classical Vibrio, was the authors observation of the temporal incompatibility of phages with acute cholera. The authors noted that only small numbers of phages were used relative to the high number of Vibrio in the intestine. The authors further noted that the transit time in an ‘actively purging’ cholera patient is sometimes as short as 20 min. Meanwhile, at least 30 min is required to complete a phage replication cycle. Effective phage-mediated elimination of Vibrio would therefore have required many rounds of phage replication over several hours, time not afforded by the action of cholera toxin. It’s therefore likely that many phages were simply excreted before they were able to undergo replication. Moreover, it was not reported whether pre-phage gastric neutralisation was performed and it is therefore unclear the extent to which gastric pH may have reduced the amount of phage progressing beyond the stomach.In 1971 Marcuk and colleagues evaluated the efficacy of oral phage therapy for the treatment of acute cholera . This trTogether the trials of phage therapy identified in this review provide reassuring data about the safety of phage therapy, affirming the findings of other recent reviews ,13. HoweNotably, despite over 100 years of clinical use, this review identified only 16 clinical or safety trials. This partly reflects the limitations of a systematic review approach: to be eligible for inclusion in this review, trials must have been indexed in full-text form in one of three online databases and published in English. This method, although thorough and appropriate for a systematic review, does not adequately account for the history of phage therapy. Many historical trials will not be indexed in online databases. Moreover, Russian and Georgian clinical trial data is generally not available to Western audiences. This creates a bias in the impression created by examination solely of reports published in Western English-speaking contexts.The evidence presented by the trials included in this review must also be viewed in the wider context of available clinical evidence, most notably observational clinical data ,12. MostNevertheless, the available clinical results paint a reassuring picture of the safety of phage therapy; in keeping with human co-evolution with phages. To the best of our knowledge there have only been 11 instances of possible clinical adverse effects to modern phage therapy, none of which were believed by the report authors to be directly attributable to phage ,52,53,54In terms of efficacy, most patients that receive phage therapy do so because they have failed antibiotic therapy. Although it is usual for such patients to continue to receive antibiotics during phage therapy, resolution of infection in the context of antibiotic resistance or tolerance in a manner that coincides with phage therapy is both compelling and consistently observed . MoreoveClinical trial evidence has become a central tenet of Western medicine, providing broad reassurance. However, it is important to acknowledge that clinical trial data is not the only form of evidence. As we have observed among the trials discussed above, phage therapy, by its dynamic and biological nature, presents inherent difficulties to the clinical trial model. First, phage preparations will require ongoing adjustment and reformulation to adapt to changing bacterial populations and resistance patterns. For example, ongoing reformulation is performed by the Eliava Institute for their pre-formulated phage cocktails. A clinical trial of a single phage is therefore likely to be uninformative, as, notwithstanding some exceptions, it will likely need to be used in combination with other phages to mitigate bacterial resistance. Likewise, a cocktail of phages subjected to a clinical trial will likely require ongoing reformulation, fundamentally altering the ‘active ingredients’. Notably, personalised phage cocktails present similarly unique challenges to the existing clinical trial model. Second, there are potentially thousands of clinically useful phages in the environment. It would be impossible to put every phage, or combination of phages, through clinical trials for just one type of infection, let alone the breadth of bacterial infections that phage could be applied to. Third, as we have seen with the trials included in this review, successful phage therapy involves the ‘Goldilocks’ constellation of getting the right amount of the right phage(s) to the right place to treat infections containing enough susceptible bacterial cells. This will be easier for some clinical indications than others, which may require a more tailored clinical approach. Consequently, demonstrating efficacy through one-size fits all clinical trial protocols may be more difficult for certain infections. Fourth, as is also evident in this review, there is much variation between existing treatment protocols. This reflects both variation in clinical phage practice and in some circumstances the tailoring of clinical phage protocols to individual patients. For example, joint infections have been successfully treated by daily administration of phage via wound catheter, a single intra-operative administration of phage or simultaneous courses of intra-articular and intravenous phage ,45,50. TRecommendations for successful phage therapy clinical trials have been published elsewhere ,62. ThisClinical and safety trials consistently demonstrate that the use of naturally occurring phage for therapy, by various routes of administration, is safe. Clinical trials also show that phage is efficacious when the right amount of the right phage(s) are delivered to the right place to treat infections containing enough susceptible bacterial cells. However, the alignment of this constellation of factors has proved tricky for previous clinical trials and, despite this and other challenges, it is hoped that future trials will deliver the compelling results much anticipated by the field. In the meantime, the evidence around the safety and efficacy of phage therapy is considered sufficiently reassuring for ongoing compassionate use when antibiotics are unable to meet clinical needs.


Increasing antibiotic resistance numbers force both scientists and politicians to tackle the problem, and preferably without any delay. The application of bacteriophages as precision therapy to treat bacterial infections, phage therapy, has received increasing attention during the last two decades. While it looks like phage therapy is here to stay, there is still a lot to do. Medicine regulatory authorities are working to deliver clear instructions to carry out phage therapy. Physicians need to get more practical experience on treatments with phages. In this opinion article I try to place phage therapy in the context of the health care system and state that the use phages for precision treatments will require a seamless chain of events from the patient to the phage therapy laboratory to allow for the immediate application of phages therapeutically. It is not likely that phages will replace antibiotics, however, they will be valuable in the treatment of infections caused by multidrug resistant bacteria. Antibiotics will nevertheless remain the main treatment for a majority of infections. Yersinia pestis [Bubonic plague, pertussis, diphtheria, pulmonary consumption or tuberculosis, typhoid and cholera are still ominous names of lethal infectious diseases caused by diverse bacterial pathogens. Prior to the invention of antibiotics, bacterial infections killed humans irrespective of age or sex . Often ta pestis ,3,4. Sina pestis . This tra pestis . The avaa pestis .Already in the 1950s, more and more bacteria resistant to the earliest used antibiotics were isolated from patient samples. The inventor of penicillin and Nobel prize laureate Sir Alexander Fleming warned in his Nobel-seminar in 1945 that bacteria will develop resistance to antibiotics, especially if the treatment regime is undersized . Thus, vHumans live in a world where there are bacteria everywhere, and it is impossible to avoid encountering them. Most of the bacteria cause no harm to humans, while some of the bacteria are symbiotic and thereby even beneficial and necessary for the wellbeing of them. On the other hand, some bacteria have specialized to use the human body as their source of vital nutrients or growth media. This has become possible for the bacteria during the evolution through the acquisition of special virulence or pathogenicity factors that enable the specialized pathogens to defend against or evade the immune defense mechanisms of the human body, and thereby the pathogens can thus colonize the humans . DiffereYersinia pestis and Shigellae, respectively, are contagious in very small numbers. The pathogens also differ based on their infective doses. For some bacteria it is very low, even one single bacterium may be enough to start the infection; however, for other bacteria the dose may be high, even millions of bacteria . For exaSince the causative agents of bacterial infections are highly diverse, their prevention and treatment also requires diverse actions. The spreading of some bacterial infections can be prevented by improved hygiene and better living standards, therefore, a functional waste management system and the availability of clean water plays a central role. The improvement of these two factors may specifically decrease the number of intestinal infections. In addition, malnourishment of children poses an increased risk to infections. Corynebacterium diphtheria or Clostridium tetani provide protection against diphtheria or tetanus, respectively, or the virulence factors of Bordetella pertussis protect against pertussis.Vaccination is an efficient measure to prevent bacterial infections caused by pathogens that demonstrate little antigenic variability between the bacterial isolates. Such pathogens are, for example, those included in the current vaccination programs. The vaccine containing the weakened toxins produced by Haemophilus influenza bacteria that causes respiratory and other severe infections. There are also vaccines against the causative agents of tuberculosis, bubonic plague, cholera, typhoid, tularemia and anthrax, to mention but a few. On the other hand, the production of vaccines against many other pathogens has not been successful. This may be due to the nature of the infection or the fact that the bacterial species in question has a vast number, even hundreds, of antigenic variants, making the production of vaccines not feasible.The vaccination program also includes a vaccine against pneumococci that is a result of a compromise, as there are over 100 known capsule types among pneumococcus strains. Among those the 23 most common capsule types that cause human infections have been chosen . To prevClostridioides difficile, and can cause very severe symptoms. FMT may revert the normal balance of the gut microbiome and eradicate C. difficile. It has been reported that FMT may also be a viable alternative to treat disturbations of the gut caused by other bacteria. Analogously, vaginal infections have been addressed by vaginal microbiota transplantations [Prebiotics and synbiotics have been proposed as a possible solution to manage intestinal infections . Probiotntations . An intentations . This im31) of phages in the world, a 10-fold excess over the estimated number of bacteria [Bacteriophages (phages) are viruses that are specialized to infect bacteria. It has been estimated that there is an unbelievable number , they will be released from the bacterial cell by a specifically dedicated lysis system composed of lysins and holins. After receiving an exactly timed activation signal, the lysis system breaks the cell membranes and the cell wall of the bacterium, thereby releasing the phage progeny to the environment . This al−10 to 10−9 per nucleotide per generation for bacteria [A continuous arms race between bacteria and phages is going on in nature . The bacbacteria . Thus, abacteria . The sitIt is possible that during phage therapy, virulent phage-resistant mutants are selected, as was the situation in the Tom Patterson case . In such23 phage infections occur every second [4/mL or g [In nature, phages respond to bacterial defense strategies flexibly by changes that retain their infectivity. There is a consensus that one role of the phages in nature is to keep a balance between bacterial populations to prevent uncontrolled increases in the bacterial numbers. It has been estimated that 10y second . It has /mL or g ,29. This/mL or g . This wa/mL or g . This ma/mL or g ,31.As a consequence of the arms race between the bacteria and phages, the phage populations have been divided into subtypes that are specialized to certain subtypes of the bacteria. Therefore, the bacterial strains are classified as phage-sensitive and phage-resistant. This emergence of subtypes has proceeded such that within a single bacterial species there are only a few strains that are sensitive to a certain phage. Because of this, phages are typically regarded to possess a narrow host range. In practice, this means that in phage therapy each patient isolate has to be tested for suitable phages hopefully present in the laboratory phage collection, and that phage therapy is always precision medicine where the phages eliminate from the patients only those bacteria that are sensitive to the phage. The beneficial microbiome of the body will not be affected at all by the phages .Vibrio cholera [Bacteriophages have been known of for 100 years. The first observations of phage activity were reported by British researcher Ernest Hanbury Hankin in 1896 when he studied the influence of the water of the River Ganges on  cholera . The wat cholera ,35. d’HeShigella, Salmonella, Escherichia, Enterococcus, Proteus, Staphylococcus and Pseudomonas. In Georgia and Russia, physicians can use phages in routine treatment of bacterial infections. This takes place specifically when the infection is not responsive to antibiotic treatment or if the patient is allergic to antibiotics.d’Herelle and Georgi Eliava founded a phage-institute in Tbilisi, Georgia in 1923. The institute started to produce phage preparates for diverse indications, and that activity still continues in Tbilisi . Phage pWhile phage therapy is experiencing a rebirth in Western countries, the present legal issues on medicinal products places special requirements on phage therapy and phage products. This regulatory framework is not completely clear yet ,39. The Phage therapy as precision medicine implies that the causative agent of the infection is identified and cultured so that it can be tested for phage susceptibility.The interaction of a phage with the target pathogen may differ dramatically in vitro and in vivo in the body fluids, and that can only be tested using surrogate systems.To meet the demand, the phage therapy laboratory needs to possess a phage collection that covers all the most common pathogens. This could involve several thousand phages, the management of which requires skilled personnel and dedicated laboratory space. All the phages need to go through a thorough characterization before they can be approved for phage therapy Production of safe phage products, even at a small personalized treatment scale, requires that approved quality control measures are in place. The sterility, stability, and the absence of endo- and exotoxins or other harmful compounds has to be ensured. In future, it is likely that phage products will be, at least partly, produced in dedicated GMP-facilitiesWhile natural phages are relatively easy to isolate against many pathogens, there are still many bacterial species for which phages are rare or even non-existentWork on phages against anaerobic bacteria or BSL class 3 pathogens presents additional challengesA realistic view is that phage therapy is not a magic bullet that would be “The Cure of Everything”. It has its inherent limitations:In the context of phages, we also need to discuss (i) the (in)direct utilization of basic phage products as potential actors in therapeutic purposes, and (ii) the engineering of wild phages to better meet the need of therapeutic phages. With respect to the use of the phage products, endolysins have received most attention. Endolysins, with the help of holins, break down the bacterial cell wall and release the phage progeny to the environment at the end of the phage life cycle ,42. As eMost phages encode products that take over the bacterial systems immediately after the injection of the phage genome into the bacterial cytoplasm. A detailed understanding of these effectors would likely open possibilities to develop drugs to their targets .7 variants [In many cases, suitable phages for patient isolates are not found, and then the question of the generation and production of recombinant or even synthetic phages becomes actual. A scenario for the year 2035 on this was recently presented . It is cvariants .Mycobacterium phages converted the phages into lytic ones that were subsequently used in the treatment of a lung transplantation patient who suffered from a Mycobacterium abscessus infection [Finally, in cases where suitable lytic phages are not available for a specific pathogen but temperate ones exist, DNA engineering can be utilized the delete the gene(s) involved in directing the phage to the lysogenic cycle. Thus, for example, the deletion of the repressor gene of two temperate nfection .While phage therapy is in active use to treat bacterial infections under diverse indications in Russia and in Georgia strongly reflecting the historical situation, it has also been actively applied in Poland at the Hirzfeld Institute of Immunology and Experimental Therapy located in Wroclaw. There, starting in the 1950s, hundreds of patients have been treated successfully, with a cure rate close to 40% ,50,51. AAfter 2000, increasing numbers of scientific reports have been published on experimental therapy trials that have used phages to cure infections caused by multidrug resistant (MDR) bacteria when the regular treatments have been unsuccessful. Typically, these treatments have not been conducted singly with phages, but phages have been combined with conventional treatments ,56,57,58At the same time a few phase I and II clinical double-blind trials have been carried out with variable success ,59. Due Increasing the therapeutic phage collection. It is recognized that the most dangerous bacterial pathogens for humans belong to the so called ESKAPEE-bacteria  that oftentimes show resistance towards most antibiotics, which makes the infections caused by them difficult to cure. Therefore, our primary aim is to isolate phages against these bacterial species. Phages are relatively easy to isolate; they are especially abundant and diverse in the sewage of cities and hospitals. Our phage collection at present comprises ca. 600 different phages. We have estimated that a collection of 1500–2000 different phages could cover 70–80% of the multidrug-resistant patient isolates. The isolated phages need to be thoroughly characterized before they can be included in the therapeutic phage collection [llection . This reRapid phage typing method. Phage therapy in the future will be precision medicine for which there are selected phages that are able to infect and kill the pathogen that has caused the infection. In practice, we screen our phage collection for suitable phages using a rapid method that also allows us to react to acute bacterial infections. To this end, we have set up a method based on liquid culture that can detect phage sensitivity in 3–4 h (unpublished results).Production of phage preparates. One of the important goals has been to set up the processes to produce and purify phages for treatment. The purity requirements of the phage products are the same as those for other medical drugs, and there are border-values for possible contaminating impurities that should not be exceeded in the final preparate. We have optimized a purification process that requires no harmful chemicals and that can be scaled up upon request. For that we systematically tested several purification protocols in different combinations and the results were published in an article that raised wide interest [interest . In the In my laboratory at the University of Helsinki, we have worked since 2013 with an aim to make phage therapy possible in Finland. The work has been carried out mainly via external funding but also with support from the Faculty of Medicine, University of Helsinki, and from the Helsinki University Hospital laboratory diagnostics. We are close to being ready to offer safe and efficient phage products to treat severe bacterial infections. The following subprojects have paved the way for this:With our expertise and experience, we have prepared phage products for the treatments of ten patients suffering from different chronic infections caused by MDR pathogens. In all cases, the phage therapy has been safe, and for most patients the symptoms have been at least temporarily relieved (unpublished).While it looks like phage therapy is here to stay, there is still a lot to do. Clear instructions for actions and permissions for the treatments should be delivered by the medical authorities, and not just for experimental therapies. More practical experience on treatments should be collected, especially by clinicians that carry out the treatments. The curriculum of medical students does not include phage therapy yet, although it should be implemented there. Therefore, it is not imminent that phage therapy would become a common practice in the near future. Upon the accumulation of experience, knowledge of phage therapy will spread among physicians and among patients, and this will promote the position of phage therapy as a seriously considered alternative in the treatment of bacterial infections. Phages most likely will never replace antibiotics completely; however, they will be valuable in the treatment of infections caused by multidrug resistant bacteria. Antibiotics will still remain the main treatment for the majority of infections, especially the acute ones, for a long time.In principle, phage therapy could in the future target any bacterial infection irrespective of whether the causative pathogen is sensitive or resistant to antibiotics as long as suitable phages are found for the pathogen. In practice, the use of precision treatments will require a seamless chain of events where the bacterial pathogen isolated from a patient sample, especially if it is MDR, will be transferred to a phage laboratory that screens the phage collection for suitable phages. In the best case, suitable phages can be ready-made products on the storage shelves. In such a case, a phage cocktail can be delivered to the clinician within a single working day. If the phage is not ready and immediately available, production and purification of the phage will take at least three to four days, to which a few days needs to be added for quality control. In the case that no phages are found in our collection, there is a possibility to get such from other phage laboratories around the world. The patient isolate can be shipped abroad in a couple of days and tested for suitable phages in the other collections. Finally, if this also fails, there is always a possibility to isolate phages from sewage samples. These last possibilities take from several days to weeks, so for the treatment of acute infections, the only possibility is that a suitable phage is already available.


Pseudomonas aeruginosa or Escherichia coli—two VAP-associated pathogens—in naïve mice without the confounding effects of a bacterial infection. Active or UV-inactivated phage cocktails or buffers were injected intraperitoneally daily for 7 days in C57BL/6J wild-type mice. Blood cell analysis, flow cytometry analysis, assessment of phage distribution and histopathological analysis of spleens were performed at 6 h, 10 days and 21 days after treatment start. Phages reached the lungs and although the phage cocktails were slightly immunogenic, phage injections were well tolerated without obvious adverse effects. No signs of activation of innate or adaptive immune cells were observed; however, both active phage cocktails elicited a minimal humoral response with secretion of phage-specific antibodies. Our findings show that even repetitive injections lead only to a minimal innate and adaptive immune response in naïve mice and suggest that systemic phage treatment is thus potentially suitable for treating bacterial lung infections.Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to two different phage cocktails targeting either  Pseudomonas aeruginosa (P. aeruginosa) or Escherichia coli (E. coli), which are on the WHO list of “global priority pathogens” continuously developing antibiotic resistance [Given the rising incidence of multidrug-resistant (MDR) bacterial pathogens, bacteriophages (phages) are becoming a focus of interest for treating infectious diseases ,2. Ventisistance ,6.An increasing number of in vivo studies in animal models ,8,9, as Although the interaction of phages with the innate immune system is not fully understood yet, natural or therapy-induced immunity against therapeutic phages remains a potential concern. Since phages are, among others, an ubiquitous part of the microbiome ,26, theyP. aeruginosa, highlighting another potential advantage of phage therapy against MDR-bacteria [In addition to a phage-specific immune response, a significant risk of phage therapy is the development of bacterial phage resistance . To redubacteria .P. aeruginosa or E. coli, two clinically highly relevant MDR-pathogens responsible for a large number of VAP cases. To avoid the confounding effects of phages infecting bacteria and pre-existing activation of the immune system by bacterial breakdown products or the bacterial infection itself, we assessed intraperitoneal phage administration in uninfected, naïve mice. Although the two phage cocktails were slightly immunogenic, even repetitive injections over 7 days led only to a minimal innate and adaptive immune response in naïve mice at either 6 h or up to 21 days after treatment start. The two phage cocktails were well-tolerated and disseminated throughout the whole organism, including the lungs. These data potentially open the way for further investigations towards the efficacy of the systemic administration of phages to treat MDR-bacteria-induced VAP.Although it was recently shown that the success of pulmonary phage therapy relies on the synergistic action between phages and neutrophils , the immAll animal studies were approved by the institutional and local governmental authorities at the Charité–Universitätsmedizin Berlin and Landesamt für Gesundheit und Soziales (LaGeSo) Berlin.Mice were housed under specific pathogen-free conditions with free access to food and water and a 12 h (h) light/dark cycle. Animal housing and experimental procedures complied with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines and recommendations for the care and use of laboratory animals.P. aeruginosa cocktail 5 × 107 PFU/injection per phage, anti-E. coli cocktail 1 × 108 PFU/injection per phage), UV-inactivated phage cocktail or buffer  buffer ) once or daily for 7 days (d). The mice were monitored every 24 h for body temperature, body weight and general condition according to pre-defined score sheets. At 6 h (n = 9 per group), 10 d (n = 9 per group) or 21 d (n = 5–6 per group), mice were euthanized with ketamine (200 mg/kg body weight) and xylazine (20 mg/kg body weight). At the 6 h and 10 d time points, intraperitoneal lavage and bronchoalveolar lavage (BAL) were performed, blood samples collected and lungs and secondary lymphoid organs  removed for further analysis. Blood cell analysis, flow cytometry analysis and determination of plaque-forming units (PFU) were performed. At the 21 d time point, blood samples were collected and spleens were removed for histopathological analysis.The 8–10 week old female C57BL/6J WT mice , a common model organism in preclinical research, were used. Mice were injected intraperitoneally (i. p.) with 100 µL of either active phage cocktail . In addition, whole blood was centrifuged at 15,000× TM, Mini Protease Inhibitor Cocktail; Roche, Basel, Switzerland). BAL and peritoneal lavage samples were stored on ice and analyzed for PFUs.Mice were subjected to deep anesthesia. Peritoneal lavage was performed by flushing the peritoneal cavity (PC) with 5 mL 1 × phosphate-buffered saline (PBS). After exsanguination via the vena cava, the tracheas were cannulated and lungs lavaged twice with 0.8 mL 1 × PBS protease inhibitor (PI) solution . The homogenates were stored on ice for PFU analysis.Lavaged lungs were perfused via the right heart chamber with 10 mL 1 × PBS and homogenized in 1 mL 1 × PBS PI-solution with gentleMACSSpleens were removed and stored in 1 × PBS on ice. PFU determination was performed with the supernatant of the single-cell suspensions described below. Draining lymph nodes  were collected, pooled in 1 mL PBS and stored on ice for further use.g for 5 min at 4 °C), the supernatant discarded and the pellet resuspended in 2 mL 1 × red blood cell lysis buffer  for 2 min (spleen only). Cells were washed, centrifuged, pellets resuspended in PBS/0.1% BSA buffer and analyzed by flow cytometry.For isolation of leukocytes from spleen and lymph nodes, the tissue was first forced through a 70-µm cell strainer  using a syringe plunger. After washing the cell strainer with 1 × PBS, the resulting cell suspension was centrifuged  and stained with anti-CD45 , anti-CD11c , anti-CD11b , anti-F4/80 , anti-Ly6G , anti-MHCII , anti-CD80  and anti-CD86  monoclonal antibodies (mAbs). The gating strategy for the analysis of innate immune cells is shown in For analysis of adaptive immune cells, cells were blocked with anti-CD16/CD32 , surface stained with anti-CD3 , anti-CD4 , anti-CD8α , anti-CD44  and anti-γδTCR , fixed and permeabilized with FoxP3/Transcription Factor Staining Buffer Set  and stained intranuclearly with anti-FoxP3 , anti-RORγt , and anti-T-bet  mAbs. The gating strategy for the analysis of adaptive immune cells is shown in All stained cells were analyzed using the BD FACS Canto II with the BD FACSDiva software. CountBright Absolute Counting Beads  were used for calculation of total cell numbers.The spleens of mice at 21 d were carefully removed and fixed in 4% buffered formaldehyde solution for 24–48 h, embedded in paraffin and cut into 2 μm sections. After routine dewaxing and dehydration, sections were stained with GL7  mAb and hematoxylin. Histopathological analysis of the germinal center B cells was performed by the Institute of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universität Berlin, Germany. The forming of germinal centers was scored into non-existent (0), minimal (1), low-grade (2), moderate (3) and intense (4). Section analysis was performed in a blinded manner.P. aeruginosa phage cocktail used for these experiments were prepared from plates with confluent lysis for each individual phage. After resuspension in phage buffer, centrifugation at 1254× g for 15 min w/o break, phage suspensions were filtered consecutively with 0.45 µm and 0.22 µm and stored at 4 °C until further use. Flat-bottom 96-well plates (Sarstedt) were coated with the anti-E. coli phage cocktail (1 × 108 PFU/phage) in 100 μL/well or with the anti-P. aeruginosa cocktail (5 × 107 PFU/phage in 100 µL) overnight at 4 °C. Wells were washed 5 times with PBS with 0.05% Tween 20  and blocked with 1% bovine serum albumin  in PBS (100 µL/well) at room temperature (RT) for 45 min. Plates were washed 5 times with PBS/0.05% Tween 20. Diluted plasma samples in duplicates were added to the wells (100 μL/well) and incubated at 37 °C for 2 h. Samples were diluted as follows: 1:10 for plasma IgM, IgA and 1:500 for plasma IgG testing for the active phage-cocktail treated mice, while 1:10 each for the UV-inactive cocktail groups and the buffer controls. After washing the plates 5 times with PBS/0.05% Tween 20, 100 µL/well of the corresponding detection antibodies were added and the plates were incubated for 1 h at RT in the dark. The following secondary antibodies were used: biotinylated goat anti-mouse IgG , biotinylated rabbit anti-mouse IgM , biotinylated rabbit anti-mouse IgA . The antibody solution was removed and the plates were washed 5 times with PBS/0.05% Tween 20. HRP-conjugated streptavidin solution  was incubated at RT in the dark for 1 h (100 µL/well). As a substrate for peroxidase, we used TMB . After incubating the plates for 15 min at RT in the dark, 50 µL of 2N H2SO4 (Carl Roth) was added to stop the reaction. The absorbance was measured at 450 nm using a MultiskanTM FC photometer  and analyzed with SkanIt Software 4.1 for Microplate Readers RE, ver. 4.1.0.43 and normalized by subtracting the blanks (PBS instead of plasma).Specific anti-phage antibodies  in plasma were measured by ELISA. The anti-Soluble cytokines and chemokines in plasma samples of the 6 h, 10 d and 21 d mice were measured using the Mouse Anti-Virus Response Panel  and the corresponding software provided by BioLegend. Samples were analyzed once, according to the manufacturer’s recommendations.P. aeruginosa or E. coli were used (P. aeruginosa phage cocktail includes the two Pseudomonas phages DSM 19872 (JG005) [7 PFU/phage in 100 µL) provided by the DSMZ  and ITEM Fraunhofer . The anti-E. coli phage cocktail includes the five E. coli phages 536_P3, CLB_P2 [8 PFU/phage in 100 µL) isolated, characterized and provided by Institut Pasteur . Single phage suspensions were highly purified and endotoxins were removed by the providing institutions. UV-inactivation was performed using a UVC 500 Ultraviolet Crosslinker  for 6 h at 95,000 µJ/cm2. Phages were stored at 4 °C. PFU analysis was performed with the E. coli strain AN33  and the P. aeruginosa strain DSM 107574 (PA74)   and DSM  (JG005)  . The lyE. coli phages) or spot tests (anti-P. aeruginosa phages).Blood and organ samples were serially diluted (1:10) in phage buffer shortly before performing the plaque assays, either by double agar overlay assays (anti-600 = 0.05–0.08) and cultured with shaking  until reaching early logarithmic phase . Low melting agar (soft agar) (4 mL/glass tube) was melted in a heating block (110 °C for 10 min) and cooled down to 48 °C. For the double agar overlay assay, 100 µL of the sample dilution and 100 µL of bacterial suspension were added to the liquid soft agar (top), gently mixed and poured on agar plates (bottom). For the spot test, only 100 µL bacterial suspension was added to the liquid soft agar. Subsequently, sample dilutions were spotted (4 µL/spot) in triplicate directly on the plates. Agar plates were incubated overnight  before calculating PFU/mL. The detection limit of the overlay assay for the E. coli phages against AN33 was 10 PFU/mL and the detection limit of the triplicates from the spot test for the P. aeruginosa phages against PA74 was 83 PFU/mL. Bacteria cryostocks were streaked on blood agar plates (PA74) or lysogeny broth (LB) agar plates (AN33) one day before. Bacterial cultures (tryptic soy broth (TSB), PA74; LB, AN33) were prepared (20 mL) with single colonies .The two phage cocktails  were analyzed via transmission electron microscopy by the Core Facility for Electron Microscopy of the Charité Berlin. For the negative staining, carbon-coated mesh grids were hydrophilized with Alcian blue solution (1% in 1% acetic acid) followed by washing steps in dHData are expressed as mean ± SD. For grouped analyses, two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was performed. Results were considered significant if P was less than 0.05. Significance levels are indicated in the figures. Statistical analysis was performed using GraphPad Prism 9 . Sample sizes of individual groups are indicated in the figure legends.P. aeruginosa (5 × 107 PFU/phage in 100 µL) and the second includes five phages targeting E. coli (1 × 108 PFU/phage in 100 µL).In this study, we evaluated the immunogenicity of two intraperitoneally (i. p.) injected phage cocktails in naïve mice. The first cocktail includes two phages targeting P. aeruginosa and anti-E. coli phages in all analyzed organs and compartments , except for the alveolar spaces (BAL). Phages in the BAL were only detected in one of nine (anti-P. aeruginosa phages) or five of nine (anti-E. coli phages) mice at this time point , BAL (n = 1) and lungs (n = 3) 10 days following treatment initiation  and i. p. lavage fluid ) were collected at either 6 h after the first injection or 72 h after the last injection (10 d) a and plame point b. On dayitiation b.To check whether mice showed clinical signs of illness related to the phage treatment, we monitored them for alteration in body temperature c, left aTaken together, these data indicate that i. p. injection allows for phage dissemination via the bloodstream as early as 6 h after application. Most importantly, phage treatment was well-tolerated without obvious adverse effects.To evaluate the systemic activation and sustainment of innate immunity triggered by the phage cocktails, we carried out complete blood counts at 6 h, 10 d and 21 d after start of treatment .P. aeruginosa and anti-E. coli phage cocktail treatments at this time point  and hemoglobin (HGB), were inconspicuous and did not differ between treatment groups and controls . Specifime point a. Howevet change b. At thet change a,b.To examine the adaptive immunity in lymphoid organs, which is initiated by activated antigen-presenting cells (APCs) loaded with antigen, we first probed spleens and draining lymph nodes for the presence of activated APCs in response to active phage cocktail encounter. Using flow cytometry for expression of costimulatory molecules and MHCII, we did not observe a significant increase in either PMNs, macrophages or dendritic cells (DCs) in the spleen at 6 h and 10 d a–c. PMN As DCs are the most competent APCs ,51, we tMatching measurements of draining lymph nodes confirmed the absence of innate immune activation of DCs in secondary lymphoid organs at all time points analyzed .P. aeruginosa phage cocktail, the proportions of CD4 T cells were moderately yet significantly increased at the expense of CD8 T cells , 40%  and 1.3%  of all T cells (CD3+) a–c. At 6 T cells a,b. We dOur analysis of the same T-cell populations in the spleen also failed to reveal evidence of T-cell proliferation . They di+), high expression levels of the activation marker CD44 (CD44hi) and intranuclear staining for the Th1-transcription factor T-bet. Only 1% of all CD4 T cells were of Th1 type and no increase in frequencies was observed in response to phage treatment at either time point  were identified by the surface expression of CD4 , soluble cytokines and chemokines were analyzed in plasma samples at 6 h, 10 d and 21 d after the first injection  or UV-inactivated anti-E. coli phage cocktail (III) did not show any specific positive signals. In contrast, both the active phage cocktail groups  showed minimal to minor positive signals for germinal center B cells stained by GL7 , UV-inactivated anti-d by GL7 . These tThe observed formation of minimal splenic germinal centers prompted us to further investigate the humoral immune response against the two phage cocktails. To this end, we measured phage-specific antibodies  in plasma by ELISA .E. coli phage cocktail induced an increase in IgG, IgM as well as IgA by 21 days compared to the buffer or UV-inactivated control, which for IgG was already evident by 10 d. In contrast, the treatment with the active anti-P. aeruginosa phage cocktail only induced a marked increase in IgG and only at the 21 d time point, but this did not reach significance. A small increase in IgG was also induced by the UV-inactivated phage cocktails but may be reflective of individual outliers. These findings suggest that the repetitive injection of each of the two phage cocktails initiated a minimal humoral B-cell response with secretion of phage-specific antibodies.In congruence with the presence of germinal centers, the active anti-For evaluating the immunogenicity of the two phage cocktails, we assumed that the immune response against the UV-inactivated and the active phages would be comparable, as we expected the UV treatment to merely cross-link phage DNA . To undeP. aeruginosa or E. coli in naïve mice without the confounding effects of a bacterial infection. Our results show that phages reached the lungs after systemic injection. Even repetitive exposure to the phage cocktails led to only a minimal innate and adaptive immune response, while all mice remained healthy without evidence of any adverse effects.Phage treatment of bacterial lung infections may be developing into an effective resource in the fight against the increasing antibiotic resistance of clinically relevant pathogens. As natural or therapy-induced immunity against therapeutic phages remains a safety and efficacy concern, in this study we assessed the immunogenicity of two phage cocktails targeting either P. aeruginosa phage cocktail. There was no upregulation of MHCII or co-stimulatory molecules on DCs that would indicate maturation into APCs and only a minimal pro-inflammatory cytokine profile at this time point. Despite the lack of evidence for an early innate response, we did observe a minimal adaptive immune response. Although we failed to observe the activation of main T cells or sub-populations at 10 days post-treatment, the histopathology analysis at 21 days nonetheless revealed a minimal to low-grade germinal center formation with increasing levels of phage-specific antibodies in plasma. The active anti-E. coli cocktail induced IgG and lower levels of IgA already at 10 days and IgM at 21 days, while the anti-P. aeruginosa phages induced only IgG and only at the late point of 21 days.Assessing the possible immune responses towards the two different phage cocktails, we did not observe a significant activation of innate immune cells in blood or lymphoid organs at 6 h after treatment, except for splenic PMNs in response to the active anti-A. baumannii infection study [A. baumannii phages, where intraperitoneal application resulted in the most pronounced, yet still low, immune response compared to oral or intranasal application [Phages can induce pro- and anti-inflammatory responses, depending on phage type, the route and duration of administration and the overall amount of phage virions present in the organism ,59. Conson study  or afteron study . Differelication .A. baumannii phage cocktail [Our results of increasing IgG titers and decreasing IgM titers over time are consistent with an antibody class switch. Similar findings, as well as weaker systemic IgA titers at later time points, have been shown by other studies ,33. IgA cocktail .A. baumannii-specific phage cocktail in naïve mice, this did in fact not negatively affect the efficiency of the phages in a wound infection model [Pseudomonas phages [P. aeruginosa phages belonged to the myovirus morphology, as well as four E. coli phages, the fifth belonging to the podovirus morphotype. In addition, our findings, as well as those of other groups [Although we did find some evidence of a weak antibody response to our phage cocktails, antibodies do not necessarily have neutralizing capacity—the main concern regarding the immune response to therapeutic phages—since phage-specific neutralizing antibodies could diminish therapeutic success. However, although the presence of phage-specific antibodies with neutralizing capacity, in particular IgG2a and IgG2b, was detected after repeated intraperitoneal applications of an on model . Antibodon model ,59,67. Ion model , the T4 on model ,68, as ws phages  as highlr groups ,37 alignr groups . Thus, tr groups . Whetherr groups ,33,34 anr groups ,37,38,70Pseudomonas phages [Staphylococcus phages [E. coli phage cocktail we observed in the present study.Natural antibodies result from continuous contact with endogenous phages, which make up a significant part of the mammalian virome ,71. If ss phages  as well s phages  in the ss phages . Converss phages . PrimingE. coli cocktail consisted of five different phages each at 1 × 108 PFU/phage in 100 µL, while the P. aeruginosa phage cocktail contained only two each at 5 × 107 PFU/phage in 100 µL, resulting in a higher total phage sum applied for the E. coli cocktail. Endotoxins and other bacterial components that remain in the solution during phage production may elicit immune responses by themselves [E. coli phages had a lower endotoxin level compared to the anti-P. aeruginosa phage cocktail, indicating that in our study endotoxin concentration did not correlate with immunogenicity. Despite the two different protocols, the immune responses to each phage cocktail remain globally weak showing that different processes can lead to similar outcomes. The absence of humoral immunity observed with UV-inactivated phages is elucidated by the EM analysis. It revealed that the UV-inactivation protocol led to major damage to viral particles. Thus, intact phages seem to elicit minimal humoral-immune responses, while phage fragments alone seem non-immunogenic.However, the different responses to the different phage cocktails may not root in the immunogenicity of the phages themselves but rather in technical aspects: the emselves . NotablyE. coli strains, the rapid lysis of bacteria by phages did not increase the innate inflammatory response compared to that observed after antibiotic treatment [Our study has some limitations. Since we chose three different time points to study the possible early innate and late adaptive immune responses, we might have missed some responses that occurred in between. We also did not extend our analysis beyond 3 weeks, which may have provided insight into the later humoral response. In addition, we did not investigate B- and T-cell memory, which are the main parameters of rapid induction of phage-specific antibodies following phage treatment after a second interval. Moreover, our study is limited to the two phage cocktails investigated here with their corresponding phage concentration. Preparations with lower or higher phage content might induce differential immune responses. In our study, we used naïve mice under SPF conditions without a bacterial infection, to avoid any confounding immune effects by the infection itself. However, the presence of bacteria may indirectly affect the immunogenicity of phages as well. Phage-induced bacterial lysis can further boost inflammation via bacterial particles and PAMPs, as well as cell-free phage DNA and higher numbers of phage virions released locally , potentireatment . Neverthreatment .In the context of lung infections with MDR-bacteria, phage therapy is of great interest for treating VAP patients. While recent studies used intratracheal applications  or nebul


Pharmacodynamics of phages mainly focuses on the phage titer used to kill the infection-causing bacterial cells; thus, a number of phages or phage cocktail standardization are very important before administration or initiation of phage therapy. In addition, the phage solution must be free from any type of pyrogenic substances that could cause an altered immune response. Pharmacologically, phages can be treated as very selective and specific toxic antibacterial agents  epithelial barrier circumvention via a Trojan horse mechanism in which phages hide inside bacteria, 3) direct sampling of luminal material by intestinal dendritic cells, and 4) phage translocation across a damaged epithelial barrier (Antigen-presenting cells (APCs) must process and present phages to T cells to trigger the generation of antibodies (Abs) and produce a long-lasting memory response. Several mechanistic theories have been proposed to explain how phages manage to get past epithelial barriers and into the generally sterile lymphoid organs: 1) phage particle transcytosis  barrier . The pre barrier . Phage-mThe present research topic focused on phages, which has highlighted the current immunology and pharmacology of phage therapy along with its related advancements. This issue comprised a detailed case report, opinion, and a review article on bacteriophages.Gondil and Chhibber, 2021 focused on the encapsulation system of bacteriophage and endolysin, which is found to be a promising strategy to improve therapeutic outcomes of phage therapy. Phage and endolysins’ encapsulation can improve various pharmacokinetic parameters with improved host immune response. The authors systematically discussed the role and advantages of endolysins (phage-borne lytic proteins) such as improved specificity, modular structure, rapid host lysis, and reduced chances of resistance. The authors mentioned various advanced encapsulation systems such as natural polymers, synthetic polymers, liposomes, nanosphere, and electrospun fibers. Thus, this type of encapsulation for phages protects from digestive enzymes and bile juices and provides permeability to the mucous lining. However, the endolysin delivery system of phages faced more challenges than phage delivery due to its proteinaceous nature and labile enzymatic activity. Thus, organic solvents play a major role in delivering the encapsulation of phages. The authors also mentioned the challenges and benefits of the endolysin delivery system; however, phage encapsulation system has been successfully reported in many clinical applications. As reported worldwide, these novel phages and endolysin delivery systems would be a promising method of drug delivery to combat multidrug-resistant infections.Johri et al. investigated a case report of successful phage therapy against Chronic Bacterial Prostatitis (CBP) in a 33-year-old male. Before the start of the phage therapy, the patient underwent multiple courses of antibiotics treatment without any long-term clinical benefits. The case report stated the culture of prostatic secretion and semen samples with the presence of Staphylococcal species such as Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus haemolyticus, Enterococcus faecalis, and Streptococcus mitis. Thus, specific phage preparations were finalized from Eliava Institute and were administered as oral liquid, rectal suppositories, and urethral instillations. The case defined significantly decreased symptoms after the treatment period (approximately 2 months) such as high body temperature, weakness, night sweating, and chills. Thus, phage therapy can be proven to be a better alternative in CBP cases with improved clinical manifestations.Kaur et al. systematically reported the role of nanotechnology in phage encapsulation, which can be an ideal pharmaceutical formulation approach in overcoming the pharmacological barriers such as less bioavailability, low stability, targeted delivery, inactivation of active phages, poor in-vivo retention, maintenance of viability, neutralization by the immune system, and poor penetration. Instead of focusing on nano-encapsulation, the authors mentioned a detailed insight about the recent nanotechnologies such as lipid-based nano-carriers, microfluidic, surface modification, nano-emulsions with integrated microfluidics for phage-cocktails, phage-loaded nanofibers using electrospinning method, and smart drug delivery platforms to control the high phage counts as required.Thus, overall, in this alarming crisis due to multiple drug resistance microbes, the development of newer antibiotics alternatives with improved efficacy is the need of the hour. Phage therapy is one of the new rays of hope among the existing alternatives. The major limitations, such as immunological and pharmacological barriers, in front of phage therapy can be overcome by using these advanced formulation and drug targeting approaches. The immune system is one of the major challenges for scientists working on phage therapy, however lack of social acceptance due to minimal public awareness and unacceptable regulatory guidelines are the major hurdles to addressing and establishing phage therapy as one of the safest alternatives for MDR microbes and their associated infections. The aim of this research topic was to address the unique mechanism and pharmacological challenges in phage therapy. Finally, this topic offers an in-depth opinion and case report of phage therapy along with the successful delivery approaches of phages, which may inspire many researchers worldwide to continue working in or enter the science of phage preparation, formulations, and targeted delivery against various uncurable infections.


Staphylococcus aureus (S. aureus) represent a major challenge to successful treatment. Further, although bacteriophages (phages) could be an alternative to antibiotics, there exists a lack of correlation in phage susceptibility results between conventional in vitro and in vivo assays. This discrepancy may hinder the potential implementation of bacteriophage therapy. In this study, the susceptibility of twelve S. aureus strains to three commercial phage cocktails and two single phages was assessed. These S. aureus strains  were compared using four assays: the spot test, efficiency of plating (EOP), the optical density assay  and microcalorimetry in human serum. In the spot test, EOP and optical density assay, all cocktails and single phages lysed both methicillin susceptible and methicillin resistant S. aureus strains. However, there was an absence of phage-mediated lysis in high concentrations of human serum as measured using microcalorimetry. As this microcalorimetry-based assay more closely resembles in vivo conditions, we propose that microcalorimetry could be included as a useful addition to conventional assays, thereby facilitating more accurate predictions of the in vivo susceptibility of S. aureus to phages during phage selection for therapeutic purposes.Infections involving antibiotic resistant  Staphylococcus aureus (S. aureus) causes many types of infections, ranging from relatively harmless skin infections to life-threatening endocarditis. In addition, it is the leading cause of bloodstream infections in industrialized countries .,63.S. auS. aureus but also multiple other bacterial species. To examine the content of the three phage cocktails, next generation sequencing was used. Firstly, a general overview of phage diversity in the cocktails was obtained using BLASTn. This confirmed the presence of multiple bacteriophage genera that infect various bacterial hosts, including S. aureus  plate-based assays such as ‘efficiency of plating’ (EOP) [Even though conventional assays showed lysis of most of the g’ (EOP) ,64. TherS. aureus to clump together in aggregates [S. aureus aggregation. However, S. aureus grows less efficiently in human serum compared to TSB, meaning that higher starting concentrations of S. aureus were required in order to measure sufficient metabolic activity of bacteria in human serum. With a higher starting concentration of bacteria, but not a higher concentration of the phage cocktails, the MOI was automatically reduced and therefore lower in the MC compare to the OD assay. For this protocol, we assessed the susceptibility of the two most phage-sensitive clinical S. aureus strains (Mup15 and Mup2723) to the phage cocktails in heat-inactivated human serum. As a control for bacterial cell death, the antibiotics rifampicin and flucloxacillin were used. While there was a clear effect of the antibiotics, the addition of the phage cocktails did not result in a decrease of metabolic activity at an MOI of 0.03  cause gregates . Microca of 0.03 . FurtherS. aureus phages, a total of eighteen phages were isolated from the RPC. The host range of these phages was determined using the spot test (data not shown). Two phages with a broad, but not identical, host range were selected: namely, RPCSa1 and RPCSa2 , Kayvirus phages bind to the backbone of WTA and are therefore not affected by these modifications [In both the OD assay and spot test, three commercial phage cocktails and the single phages used, there was a broad host range against clinical ocktails ,68. Thes-systems ,69,70. Iications ,69,70,71S. aureus strains were lysed in our experiments. Interestingly, the genomes of the S. aureus strains that showed low phage susceptibility were not genetically closely related to each other, as shown by the phylogenetic analysis. Therefore, no clear link between phage susceptibility and the genetic background of our S. aureus strains could be made. Moreover, the evaluation of known phage-resistance genes in the genome could not explain the differences of phage susceptibility of the isolates either. For example, although Mup3199 was resistant to the phage cocktails tested, no known phage-resistance genes were found. In contrast, R5 is very phage-sensitive despite the presence of multiple phage-resistant genes. This is consistent with the observation of Moller et al. that phage susceptibility relies on both host factors, most of which are still unknown, and phage-specific factors. The lack of a clear relation between the genetic background of isolates and their phage susceptibility highlights the importance of susceptibility testing prior to treatment with phages [Despite these phage characteristics, not all h phages .S. aureus strains tested. For example, strain M116 was susceptible to GPC and RPC in the spot test but not in the OD assay. This supports previous observations that the spot test might give an overestimation of phage susceptibility [S. aureus strains that were not susceptible in the spot test showed a decrease in growth in the OD assay. However, this high MOI might not be realistic for in vivo use because bacteriophages do not accumulate well in all tissues and are cleared from the blood both passively by the spleen and liver and actively by the immune system [Comparison of the spot test and OD assay did not reveal identical phage susceptibility patterns for the tibility . Discrepe system . Some ofe system ,64. HoweS. aureus in human serum, resulting in reduced phage propagation [In contrast, MC allowed the determination of phage susceptibility in human serum, despite the presence of bacterial aggregation, showing a drastic decrease in phage susceptibility to both the phage cocktails and single phages when compared to TSB. However, it should be noted that due to the unknown content of the commercial phage cocktails used, we were not able to concentrate them without the risk of selecting for specific phages . As a repagation ,74. Howepagation ,73,75,76S. aureus. The lack of phage susceptibility in human serum could explain the discrepancy between in vitro results obtained using conventional assays and in vivo data from clinical phage therapy trials. For example, the intravenous administration of AB-SA01 (a cocktail containing Kayvirus phages) resulted in a response rate of only 62%, despite the spot test indicating complete in vitro phage susceptibility of the infecting bacterial strain [In this study, we highlight the importance of experimental conditions on the phage susceptibility of l strain . So evenHere, we propose the use of MC testing in combination with current conventional assays, for more accurate in vitro phage selection for phage therapy. This is due to the assay’s better resemblance to the microenvironment encountered by bacteria and phages in vivo. In the future, MC could be used to determine phage susceptibility in other media resembling in vivo environments. For example, it has already been shown to be a valuable tool for phage susceptibility determination in urine . MC migh


Bacteria and their predators, bacteriophages, or phages are continuously engaged in an arms race for their survival using various defense strategies. Several studies indicated that the bacterial immune arsenal towards phage is quite diverse and uses different components of the host machinery. Most studied antiphage systems are associated with phages, whose genomic matter is double-stranded-DNA. These defense mechanisms are mainly related to either the host or phage-derived proteins and other associated structures and biomolecules. Some of these strategies include DNA restriction-modification (R-M), spontaneous mutations, blocking of phage receptors, production of competitive inhibitors and extracellular matrix which prevent the entry of phage DNA into the host cytoplasm, assembly interference, abortive infection, toxin–antitoxin systems, bacterial retrons, and secondary metabolite-based replication interference. On the contrary, phages develop anti-phage resistance defense mechanisms in consortium with each of these bacterial phage resistance strategies with small fitness cost. These mechanisms allow phages to undergo their replication safely inside their bacterial host’s cytoplasm and be able to produce viable, competent, and immunologically endured progeny virions for the next generation. In this review, we highlight the major bacterial defense systems developed against their predators and some of the phage counterstrategies and suggest potential research directions. CurrentBacteria and phages are seemingly involved in a continuous battle. It is part of continuous cycles of coexistence and evolution, resulting in phage-resistant hosts protecting bacterial lineages, while counter-resistant phages threaten such strains. Phages, by developing resistance, play a crucial role in controlling bacterial populations in most, if not in all, the milieus. Bacteria can evade the phage attack via several mechanisms and some of these strategies include the following: DNA restriction-modification (R-M), spontaneous mutations, blocking of phage receptors, production of competitive inhibitors, and extracellular matrix and acquired immunity via the clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) mechanism ,7,8. On In this review, we present an overview of the major anti-phage defense strategies of bacteria and the counterstrategies used by phages to evade these systems and suggest potential research directions.The mechanism of phage resistance could take place at various stages of phage replicative cycle. During the phage replication cycle, a phage introduces its DNA via translocation into the host’s cytoplasm. This will lead to either the lysogenic cycle (prophage formation) or the lytic cycle. In the lytic cycle, phages pass via several steps and early and late gene expression, which leads to maturation and aggregation of newly produced virion, which are ultimately released via lysis. The phage resistance strategies can interfere with one of these steps as shows in Campylobacter fetus slime layer) or capsules  can cover the bacterial surface and makes the phage receptors inaccessible for phage binding so that protecting bacteria from phage attack [Campylobacter jejuni (strain NCTC11168) against phage F336. The authors proven that the successful adsorption of the phage F336 to the bacterial surface depends on the hypervariable O-methyl phosphoramidate (MeOPN) modification of capsular polysaccharides (CPS). However, phage resistance has been acquired by loss of MeOPN receptor on the surface of the organism because of cj1421 gene phase variation, which encodesd the MeOPN-GalfNAc transferase [Phage adsorption to the bacterial cell surface is performed through specific receptors as the first step of phage infection cycle. Several bacterial cell surface proteins, lipopolysaccharides, and other surface polysaccharide and carbohydrate moieties can serve as receptors for phages . Bacterie attack . Some bae attack  . In the case of Sie defense systems phage encoded anti-phage proteins can be utilized by bacterial cells to prevent the translocation of DNA of lytic phage into the cytoplasm of host cells, thereby acquiring protection against virulent phages  Figure . These pgp5, downregulate the activity of the T4 lysozyme. The activity of the T4 lysozyme encoded by gp5 has inhibited the membrane protein Sp, thereby likely preventing the peptidoglycan degradation and the consequent translocation of phage DNA [Virulent phages, such as Coliphage T4, have two Sie systems mediated by Sp and imm. These systems prevent the entry of phage DNA into the cytoplasm of bacterial cell, thereby affecting successive infection by other T-even-like phages . The Sp hage DNA ,19.Neisseria gonorrhoeae and others such as Helicobacter pylori, Streptococcus pneumonia and Haemophilus influenzae have abundant R–M systems [Restriction-modification (R-M) systems are universal and tremendously varied in the bacterial primitive immune system. The R–M system has two main components: a methyl transferase (MTase) and a restriction endonuclease (REase). The restriction endonuclease pathway detects short DNA segments, measuring between four to eight base pairs long, and they are chopped into pieces. The mis-recognition and cleavage of the host DNA is protected by the methyl transferase, which is hidden from being recognized by the restriction enzyme . The pha systems .There are four R–M systems, so far identified, based on their subunit composition and mechanism of action . Type I The genome of some virulent phages may be modified by harboring a rare base hydroxymethylcytosine (HMC) in place of the base cytosine. This modification enables T4 phage DNA to be resistant to the classical R–M-based degradation. In co-evolutionary warfare, some bacteria have developed modification-dependent systems (MDSs) that used to attack the modified DNA of phage .Staphylococcus aureus, where they are specifically located in the pathogenicity islands name as “SaPIs”. In Gram-positive bacteria, PICI expression has been downregulated by a transcription repressor. PICI is excised from the host chromosome by the anti-repressor that has been produced by helper phages. Proteins translated from PICI genome suppress the expression of late helper phage genes and also modify the size of capsid protein to be suitable to accommodate the PICI genome, which ultimately end up with proper packaging of PICI genomes and prevent the formation of helper phage virions [Phage-inducible chromosomal islands (PICIs) are one of phage resistance mechanism developed by bacteria, which involve the integration of small (∼15 kb) gene sequences and are excised with the help of a specific “helper phage” . The gen virions   is a strategy of bacterial cells halt the release of newly produced progeny virions at the expense of the life of infected cell, thereby preventing the uninfected cell from being infected. It is considered a self-sacrificing event, or apoptosis that averts the subsequent infection of the neighboring bacterial community . Each Abed cells . Prematued cells . Anotheron cycle . An Abi proteins . In gene. lactis   [E. coli can inhibit the infection process of T4 phage by inducing MazF’s ribonuclease activity, which ultimately leads to complete cessation of the infection cycle [Phages may trigger the bacterial cells to produce toxins, which my attack the phage infection cycle. There are six major bacterial toxin–antitoxin (TA) types, classified based on the toxin neutralization mechanisms, the biomolecular and functional characteristics of the TA and, the TA count  . This wion cycle . Table 1E. coli retron, Ec48, has 48 nt long reverse-transcribed DNAs [E. coli RecBCD, causing the retron (Ec48) to activate and kill the cell via Abi [Retrons are bacterial genomic materials composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). The ncRNA served as a template for RT, producing a chimeric DNA/RNA molecule in which the DNA and RNA are covalently connected . Retronsbed DNAs ). It has via Abi . The autStreptomyces spp. They reported that Streptomyces spp. synthesized two vital bioactive metabolites (doxorubicin and daunorubicin) that bind  into the DNA of phage and prevent the replication cycle. Interestingly, the bacterial growth pattern was not affected by these molecules. It has been proposed that daunorubicin exerted its action at an early stage in the phage replication cycle, after the translocation of the DNA, but ahead of replication. Doxorubicin forms free radicals , which can damage DNA and cause DNA oxidation  enzyme, which cleaves the phage DNA at specific recognition site(s). In response to this defense feature, phages developed a broad range of passive and active anti-restriction strategies ,64 Figu.Myoviridae coliphage P1 encodes two inti-restriction proteins, DarA and DarB, that are co-translocated into host cells, along with their genome, thereby hiding the type I R-M recognition sites and protecting phage DNA degradation [The radation . In addiradation ,67.In the case of passive mechanisms of phage evasion, MTase modifies the double-stranded DNA of phages rapidly within a host comprising a R–M system before it is recognized by the host REase. Thus, the invading phage DNA will be protected from degradation by R–M systems. Hence, the modified genome of phage can replicate safely in the R–M-consisting host cell and can also infect and replicate in other cells which express identical R–M system. Nevertheless, as the R–M system is specific to specific host, the same DNA of a phage can be detected as foreign in a cell consisting of a different R–M system, and it will, therefore, be degraded by a different REase. In yet another twist, some REases of bacterial cell even can detect and degrade modified DNA .Lactococcus phages are the best example for this counterstrategy. Mutations of one or more specific genes of the Lactococcus spp. can enable the phages to escape the Abi systems [Lactococcus spp., for instance, can bypass the AbiQ mechanism by mutating genes involved in nucleotide metabolism. A phage can encode a molecule (such as Dmd in coliphage T4) that can replace a bacterial antitoxin, thus inhibiting the activity of the bacterial toxin and protecting the cell from death [Pectobacterium atrosepticum phage, phiTE, produces pseudo-anti-toxin RNA or takes over the antitoxin, ToxI, during its infection to deactivate the ToxN toxin [Certain phages have developed anti-Abi mechanisms to undergo protected replication in the targeted host cell . Lactoco systems . Phages om death . It has xN toxin .An individual nucleotide substitution can enable phages to evade the CRISPR system in the protospacer site or in the conserved region adjacent to the protospacer motif . In contPseudomonas aeruginosa temperate phages [Current research findings indicated that phages encode anti-CRISPR genes, which are active towards the CRISPR of bacterial defense systems, as it is recognized in e phages . Some phe phages .Some examples of the phage’s counterstrategies towards different phage resistance mechanisms that we discussed so far are summarized in In this review, we have reviewed a variety of phage resistance mechanisms. These antiviral defense systems involved several biomolecules, proteins, enzymes, cellular structures, and inter-cellular interactions that protect bacterial cells from lysis and death. The bacterial antiphage defense strategies, which are described in this review, reflect the tremendous diversity of phages, and thus some other resistance mechanisms could be discovered.The study of phage resistance should be scaled up beyond the discovery of the baseline mechanism towards the detailed understanding on the molecular root behind these antiviral systems. In this context, advancement in phage biology will certainly be required to fully understand the mechanism of phage resistance. Moreover, most studied antiphage systems were associated with the phages whose genomic matter is double-stranded-DNA. Resistance mechanisms linked to single-stranded DNA and RNA, or double-stranded RNA genomes, as well as other genetically unique phages, are waiting to be investigated. In addition, lysogenic phage mediated resistance mechanisms are less understood, and therefore this gap needs to be filled.Siphoviridae) are not continually evaluated.The above-mentioned phage resistance strategies are often investigated in a laboratory-controlled setting, in a single replica, and using a single host–phage model. Nevertheless, bacterial pathogens usually comprised multi-lateral antiphage systems. The coexistence of these systems in one host cell has seldom been studied, and the outcome of such interactions on phage evolution and their community is often overlooked. Likewise, the effectiveness of phage resistance strategies towards some families of phages (non As bacteria and phages have ancient co-evolutionary history, phages can profoundly generate a counter-resistance, via several mechanisms and with small fitness cost. One phage may also possess multiple phage resistance systems that may generate strong defenses over individual systems, allowing specific clonal inhabitants to stay in phage-containing ecosystems. The phage counteracting strategies enable the phages to undergo their replication safely and to produce viable progeny virions for the next generation. However, there are many unexplored mechanisms, cascades of reactions, molecular interactions, and involvement of newly produced biomolecules that need additional in-depth investigation for better understanding of phage science. Hence, the limitation of this review is that we have presented limited information regarding the mechanisms of action of some phage resistance and counterstrategies that have not yet been fully explored.