Phage’s: a dissertation without a title
Introduction
The world is at a tipping point. Antimicrobial resistance has left the human race in crisis. Without intervention, the implications are likely to be devastating . We are about to enter a “post-antibiotic era,” as described by the World Health Organisation. Bacteriophages, viruses that target specific bacteria, could be the answer to the threat that is posed. With the technology known to the Western world since the start of the 20th century, why is it that they are yet to be clinically introduced?
Phages are the single greatest biological entity by mass, with an approximate 1031 existing at each moment . They account for the death of as much as 40% of bacteria on water surfaces each day . The antibacterial nature of bacteriophages was first stumbled upon in 1896 by Ernest Hankin. However, he characterised them only as an antibacterial substance that could pass through fine porcelain filters .
The first description of bacteriophages as we understand them today was made by Felix d’Herelle, after Frederick Twort’s original observation of the lytic zone around phage infections in 1915. As a result, phages were used clinically as a treatment for a group of infected children . With limited controls and great dispute about the results , the research was riddled with controversy. Despite this, D’Herelle’s research continued throughout the 20th century, finding relative success in the treatment of cholera in 1931. From an experimental group of 73 subjects, he proved a 90% decrease in deaths against the control group . Despite this, the existence of phages was still being disputed until mainstream use of electron microscopy ended the debate . Even after this, the administration of phages was limited by a lack of understanding of their characteristics.
Despite ongoing debates, the success of d’Herelle’s research led to entrepreneurs funding the production and preparation of phages. They were to be shipped across the world but were riddled with flaws that made them unreliable, leading to phage therapy’s reputation being effectively ‘debunked’ in Western medicine . This lack of reliability was affected by several factors. Low titres were caused by poor purification and lax storage procedures.
Another limitation was the lack of effective delivery systems for the phages at the time, leading to the innate immune response removing the phages from the bloodstream .
In Eastern Europe, these concerns were addressed in the Soviet era, and phage therapy is still used to this day . In the West, there is a different story.
The downfall in the Western use of phages was not simply because of a lack of reliability, but also because of the changing context of their use. A greater understanding of the role that sanitation played in disease led to a great increase in life expectancy. At the turn of the 20th century, the life expectancy in the United States (U.S.) was 47. By the 1950s, it had risen to above 70 . This correlated with the widespread use of antibiotics and a corresponding decrease in the number of deaths caused by bacterial infections .
The 1960s became a golden era of discovery and development of new antibiotics, yet since then, the focus has shifted towards the modification currently utilised antibiotics . There now only exist four multinational pharmaceutical companies that have designated divisions focusing on the development of these new antibiotic drugs . The decline in discovery is heavily linked with the lack of finance in the field . Even with the addition of new treatments, the number of approved antibiotics by the FDA is decreasing with only 6 between the years of 2010-16 .
Over 262 million antibiotic courses were prescribed in 2011. Worldwide that year, there were over 100,000 tonnes of antibiotics used in agriculture and medicine. Resistance to the most commonly used agricultural antibiotic is now present in the environment within bacterial genes .
A unique challenge presents itself to the world’s population, with deaths likely to skyrocket as a result. Over the past decade awareness has been growing, with increased reporting of the issue in Western media and even the establishment of a group called ‘Biotechs from Europe innovating Anti-Microbial Resistance’ that act as a collaboration of companies in Europe to innovate .
Even with greater public awareness, worldwide antibiotic use is still on the rise each year . Partly, this is due to varying levels of control, with patients across large parts of the world not requiring a prescription to receive antibiotics . Declared a threat to global health by WHO , antibiotic resistance is predicted to kill over 10 million people within 30 years . Already costing the US $55 billion per year, this will be a huge burden, not only on the health but also the financial well-being of the general public . The UN General Assembly said that this crisis is the most urgent global risk that the human race faces .
Already in the US, mortality rates to resistant strains of S. aureus already outnumber the combined total of tuberculosis and HIV/AIDS-related deaths .
Bacteriophages are an appealing alternative to antibiotic treatment. Interest in the development of phages is especially high in smaller BioPharma, which has led to the development of many different treatments with varying delivery systems, which can target a number of diseases. Limited level of engagement from larger BioPharma has meant a lack of funding for increasing the awareness of phage therapy in the public. This has led to an understanding about newly developed treatments must come mostly from the scientific community. Despite this lack of engagement by the larger companies, there has been an increase in the number of trials relating to the introduction of phage therapy.
Current State of Play
Despite stalled development in the Western history of phage therapy, this was not the case in the East, where phages were welcomed by Soviet scientists and are still being used across Eastern Europe. A stronghold from the Soviet era is the Tbilisi based Eliava Institute. For many years, phage therapy was a regular part of health care and the institute produced phages in both monophage and cocktail forms. The treatments were numerous, some finding great success with lytic activity against S. aureus causing susceptibility in 98% of paediatric patients . The Eliava Institute has continuously sought to legitimise phage therapy and in doing so has carried out several infamous human trials. With trials on a range of common bacterial infections, the applications have varied from therapeutic to prophylactic . Early trials in Eliava involved phage cocktails targeting a spread of species of bacteria. Even with this shotgun approach, 74% of the patients showed improvement following their treatment .
A typhoid outbreak in 1974 led to a large-scale trial of intervention in which 18,577 children underwent testing. The result of the prophylaxis was a fivefold decrease in the incidence of typhoid .
Diabetes, an increasingly common disease in which the human body loses its ability to respond to or produce insulin, can cause ulcers on patients’ feet. These can easily become infected by S. aureus. Recently, trials showed that phage therapy could lead to the clearance of the infection in all patients . Considerable data has been recorded at the centre, but as the majority is recorded in non-Western languages and does not conform to Western scientific methods of trials, the data has not been used to its full potential. More recent trials by the institute indicate the wide array of potential uses for the therapy. Recently, they have looked at the treatment of widespread diseases and their secondary infections, such as those caused by cystic fibrosis.
Animal trials
Due to the lack of clinical data on phage therapy in the West, trials have a long way to go to catch up with antibiotics. Animal models have been used to investigate the potential of phage therapy against several pathogens that affect humans.
Animal trials have proved fruitful, and phage therapy has been tested across a variety bacterium. Gut-derived sepsis as a result of P. aeruginosa, when treated led to a 66.7% survival rate in contrast to none surviving in the control group . Single strain specific phage therapy led to a 100% survival rate of mice with E. facecium that had shown vancomycin resistance . This was replicated in P. aeruginosa resistant to imipenem and lactamase producing E. coli . Using hamsters with ileocecitis caused by C. difficile, a single dose of phage therapy alongside a prophylaxis led to a survival rate of 92% compared to the death of all of those treated with clindamycin. This would occur within 96 hours of infection . When phage combinations were used to treat similar C. difficile models, the growth of the bacteria decreased significantly . Animal models with antibiotic resistance have shown an even greater range of success when treated with phage cocktails, with clearance from a spread of organs .
Clinical Status of Phage therapies
Phage therapy has been used in Eastern Europe since the early 20th century. In 1919, phage therapy was already being used to treat the pathogen Shigella dysenteriae , but the adoption in the Western world was limited due to the success of antibiotics as a treatment. Despite this, phages are still used in Poland, Russia and Georgia. However, as they were introduced a relatively long time ago, they have not passed through the same clinical routes that modern antibiotics have and the number of clinical trials using natural phages as therapy is relatively small. With the increasing threat of antibiotic resistance, the number has recently risen. Chronic leg ulcer treatment has recently been targeted for trials, with the P. aeruginosa, E. coli and P. aeruginosa acting as the targets . These have all been tolerated and have passed through to the next stage of clinical trials.
When P. aeruginosa that caused chronic otitis passed through phase II clinical trials, there was a significant decrease in the bacterial load in patients that had been treated with phage therapy .
Another clinical trial targeted chronic rhinosinusitis caused by S. aureus. This trial showed that through the use of phage therapy, this infection was cleared from the sinus of every patient .
Some trials that have shown limited efficacy as well, such as a trial for the treatment of E. coli that resulted in acute diarrhoea. Compared to the traditional treatment of zinc and oral rehydration, there were no significant improvements in the health of the children that were treated despite that the fact the bacterial load had lowered . It was postulated that the number of the mixed infections that were present were the cause of the diarrhoea rather than solely the E. coli.
Despite the mixed results, an EU funded phage trial was carried out recently. The randomized trial for the treatment of burn patients infected with P. aeruginosa was the first of its kind.
As this phage therapy is only just beginning to breach Western medicine, there is no clear pathway for the regulation of phages. As this is the case, no phage therapies have passed through the framework at this point in time . The European Medicine Agency (EMA) has recognised that this is an issue. They have called for more clinical trials to be launched so that a variation of the current regulatory framework can be established .
The development of phages also requires different skills to that of antibiotics. Although the isolation of a specific phage is quite simple , the costs of the production are not easy to predict. As a result, it is difficult to raise funding. This, alongside the unpredictable nature of the regulatory pathway, means that the cost is extremely variable.
Mode of Action and Advantages
Modalities of phage therapy
Eradication is the ultimate aim of phage therapy. This can be achieved through the use of natural phages that are adapted to target particular bacterial species.
,
Stage a) is the attachment to a susceptible bacterial cell.
Stage b) is the viral genome movements through the cell, the hijacking process and its release from the cell. There are different strategies used by phages to cause the rupture of the cell membrane. Holins, for example, are used to cause a puncture that leads to the cell’s rupture. This can occur within the space of minutes if the cell is highly metabolically active.
Natural Phage therapy
A common order used as an antibacterial virus is the caudovirales. These are viruses with a tail appendage . Tailed phages are easily discovered and are found in large abundance, especially in areas rich in bacteria such as plants and animals .
They possess the ability to act in both a bacteriostatic and bacteriolytic mode, meaning that it can stop the bacteria from reproducing and also destroy it. Naturally, bacteriophages use the bacterial cells to reproduce and in this hijacking process, they kill the cell too. This raises questions about its potential to be implemented alongside regular antibiotic treatments.
Advantages
The antibiotic crisis is not the only reason to consider the introduction of a system in which phage therapy is used as a treatment for bacterial infection. There are many advantages which make it an attractive option.
Safety
The use of antibiotics has led to many undesired reactions. Acute illness and comorbidity can affect the presentation of anaphylaxis, cardiotoxicity and other complications . Anaphylactic shock is the most common adverse effect and is seen when there is an exceptionally high concentration of the drug in the tissues and is connected to specific types of antibiotics .
It could be argued that phage therapy has a comparatively preferential safety profile. In regard to the gut microbiome, phage therapy is advantageous as it has been shown to protect the natural flora . Even in immunocompromised patients, it has been argued that the adverse effects will be limited . The therapy is not without its concerns, however. There are mixed accounts of the adverse effects of Polyphage therapy. Mouse models have shown that they can induce the increased permeability of the intestines and an adverse inflammatory response. If this is the case, it could result in the development of type 1 diabetes and Crohn’s disease . In contrast to this study, cytokine levels were shown to be steady in an alternative study following phage therapy . A lack of long term studies limits the safety profile. The short-term profile has a limited number of negative reports to the amount recorded for antibiotics.
It is worth noting that phage therapy has only recently reached the intense gaze of Western medicine. As a result, there is limited literature on the safety of phage therapy, and clinical trials have only lately taken place . This lack of clinical trials needs to be addressed if phage therapy is to be utilised in the Western world.
Specificity
The natural specificity of bacteriophages to different strains and species means that the natural flora of humans is not as widely damaged as traditional antibiotic treatment. This can be extremely specific, removing the negative impact that is associated with an alteration to the natural gut flora. There is sometimes susceptibility shown by several bacteria to a single strain of phage however . Imbalances caused by antibiotic treatment can lead to dysbiosis, with many other diseases associated with the gut microbiome .
Gut bacterial dysbiosis is associated with diseases such as asthma and obesity . More commonly, there are secondary outcomes of antibiotics, including diarrhoea as a result of C. difficile, that is related to the treatment . Phage therapy, in its limited research and understanding, has shown to have far less impact on the natural flora whilst removing target pathogens .
However, there are drawbacks to this specificity, with the narrow spectrum of targets being the primary area of concern. It cannot always be presumed that there is a narrow range of targets, and single phages have been known to lyse more than 700 different strains . There are modifications that can be undertaken in order to improve the growth conditions and isolation of broad range phages . A wider range of bacterial strains can be targeted through the use of phage cocktails, which are mixtures of different bacteriophages.
These cocktails would overcome several issues. For example, burn wounds become infected often harbour multiple strains . However, phage cocktails do hold their own logistical challenges too. Knowledge of the spread of the host bacteria would still be required to target the infection. The specificity of this cocktail approach would also limit the mass production that is seen in many antibiotics as infections can vary geographically as shown in vitro . Without this specificity, phage cocktails aimed at a species and not specific strains can still show no improvement above a placebo . Advantageously, this theory can be reversed. The specificity in local areas can be used to resource the phages that could be used for the therapy . Regulatory routes must be adaptable in the addition of specific phages to cocktail therapy without the need to carry out the full set of clinical trials.
Penetration of Biofilms
An issue faced by antibiotics is biofilm development and this can limit the efficacy of the treatment . Bacterial populations can be exposed to non-lethal levels of antibiotics due to their biofilm, leading to increased resistance. High doses of antibiotics are needed to prevent biofilm growth and yet, the biofilms are rarely removed, and the concentrations of antibiotics can lead to complications and tissue toxicity .
Phages can overcome this through enzymes that are found on the capsids. These enzymes can break down the extracellular polymeric substances on the outside of the biofilm and allow the phage access to the bacteria within the biofilm . Once the progeny is released, increased layers of the biofilm are degraded . The biofilm degradation caused by phages is particularly promising for the treatment of infections caused by medical implants, especially as a preventative treatment. Phage treatments have already been shown to clear a range of bacterially produced biofilms .
The difference in their mode of action in comparison to antibiotics means that the phages are useful against bacteria that have developed resistance. There is also the potential for their joint use with antibiotics .
The genes encoding the enzymes on the capsids, if used effectively, are promising. However, a drawback to the useful genes found in bacteriophages, is that they are contained in a section of a gene referred to as ‘moron’ elements. These elements are so called because they have more on them than previously understood. There might be a potential symbiotic nature of phages within bacteria . This would contradict the belief that their only role is as detrimental parasites to the bacteria.
Phage Cocktails
Monophage therapy is unlikely to be the answer to problems with Western medicine. Polyphage therapy instead involves the use of multiple strains of phages in a single treatment. This differs from monophage therapy, which is typically used only in experimental models . Even with the advantage of ease of preparation with monophage therapy, this is outweighed by the limited therapeutic efficacy.
Phage cocktail developments
Research into phage cocktails began as a means of improving efficacy. In the first experimental treatment using phage cocktails during a double-blinded clinical trial, a clear improvement was shown against traditional treatments. Chronic otitis caused by the P. aeruginosa was reduced by 50% as identified by the visual symptoms of the disease . This showed clear evidence of their advantages.
An issue when producing phage cocktails however, is the interference between the phages being used is the ‘depressor effect’ were the phages will not function as effectively in their therapy as they would do on their own . This can be caused by the parasitism of phages or genetic transfer between different phages . The depressor effect is not enough of a disadvantage to outweigh the benefits of the increased range of targets due to the implementation of cocktails.
There are now efforts to expand the use of phage formulations and their targets. There are also efforts to prevent phages from facing the same levels of resistance seen in antibiotics. Through both of these, the commercial and medical utility will increase.
It should be noted that despite the many advantages of phage cocktails, there are more limitation to the treatment. In comparison to monophage therapy, there will be a less concentrated dose of phages that can target that one specific bacterium. There will also be the increased competition of phages to each binding site . These are both detrimental to the treatment of the patient, yet with optimisation of the cocktails the advantages outweigh these factors.
Optimisation of Phage cocktails
Phage cocktail optimisation is being used to overcome resistance to non-optimised cocktails that have been studied in specific models. Staphylococcus aureus strains that were analysed showed a range of restriction-modification including adsorption inhibition . However, by using an optimised phage cocktail to treat mice that had a Klebsiella pneumoniae dosage administered, the treatment of a phage resistant host was successful. This was shown through the use of three phage elements in the cocktail . Molecular studies of mouse models showed that even against engineered phages, bacterial resistance can increase survival times and it requires a higher phage dose to remove them . Optimisation of cocktails will continue alongside the technology that is needed in order to implement the change.
Integrating Phages and cocktails into medicine
Phage therapy in the form of cocktails does have the ability to cure bacterial infection, yet there are areas of concern.
When producing a cocktail, the larger the selection of phages, the greater the chance that it would be used without identifying the target. Additionally, the greater the number of phages, the more commercial and medical potential that these cocktails will possess. The balance that should be weighed up in terms of its clinical use is the risk of targeting commensal bacteria. The number of phages used would have to be very high to be as efficacious as traditional antibiotics. The production of additional phages increases the manufacturing cost, and ultimately lowers the commercial validity of the product.
It may be possible to produce a cocktail that not only targets all threatening bacteria but also the possible mutations that will result from the therapy. In practice, however, more specific cocktails are more likely to be used in the short run, for a number of reasons .
Personalised phage cocktails
Phage therapy has two main variants: ready to use and custom made. In an era of personalised medicines, there is potential for phage therapy to implement new technologies already being used in order to provide more personal cocktails . This may be considerably far in the future, however, as the mapping of phage interactions is in its infancy. Alternatively, phages can be used as a means of identifying the bacteria causing an infection .
Modifiable phages are a middle ground between ready to use, and custom made. They are graded in their levels of personalisation, with some more customisable than others. In the same way that a phage bank is a viable idea; a phage cocktail bank would allow groups of treatments more suitable for the patient to be selected. The issue with a phage cocktail bank would be the storage and development costs.
In Georgia, they currently use another modifiable phage cocktail system. The system targets large groups of potential pathogens. These are specific to the site of infection, such as a burns cocktail or an intestinal option. Generally, there is a set of fixed ingredients, but the cocktail changes over time to fit the most prevalent disease strains . The adaptation of phages can expand the lifespan and decrease the cost of adjusting to phage resistance.
Resistance
Bacteriophage resistance
Bacterial immune defence is more than simply developing resistance against antibiotics. Bacteria have been present in the environment as long as bacteriophages themselves, surviving the lysing process that is associated with phages. Bacteriophages are thought to be one of the key drivers in the bacterial species that they target, working antagonistically to cause a co-evolutionary process . Similar to antibiotics is the development of resistance to phages and phage therapy by bacteria .
Several studies into bacterial resistance against bacteriophages have taken place since the appearance of phages as an alternative to antibiotics. These show that even in vitro, resistance develops . It has also been shown that phages have developed methods to overcome this resistance in order to infect targeted bacterial cell walls. With anti-CRISPR identified , it appears as though there is an arms race between phages and bacteria.
Systems within bacterial cells have been identified in their genomes such as widespread use of DISARM and BREX as other means of defending against bacteriophages . There are different methods of resistance that are shown by bacterial cells. Bacterial cell surface receptors can modify in order to prevent the attachment of phages. They can be down-regulated and shielded also .
Despite this, surface receptors are generally highly conserved and are essential in the virulence of the bacteria . As a result, other defence systems are preferential. CRISPR-Cas systems can be used in the response of adaptive immunity . The resulting resistance has a relatively limited impact on the effect of phage therapy, especially when compared to the resistance against antibiotics . This is due to the vast diversity of phages that can be used when resistance develops .
However, bacteria also defend themselves against phages using restriction-modification systems in which they use restriction enzymes and methylase to protect against the foreign DNA. Despite the frequency of resistance towards phages, phages can also overcome this resistance in a natural setting . Phages are known to produce methyltransferase as a means of defence against this the restriction-modification systems .
Bacteria continually develop their resistance to phages, sacrificing growth rates even when there are none present . Phages too can train through adaption in order to be more damaging to the bacterial colonies they are targeting . In vitro and in vivo rates may differ due to the biofilm growth and the host of the bacteria. Despite an understanding of in vivo resistance, as is so often the case with phages research is limited and more is required.
In a means to further understand the relationship between phages and bacteria, co-evolution has been heavily studied in the laboratory. The evolution of both phages and bacteria was seen in vivo during a study looking at the Flavobacterium columnare isolates that had general resistance to phages. The resistance caused an increased host range in the phages along with heightened infectivity through selection . Resistance to phages in a clinical setting varies from bacterial species, which is known to be as high as 85% in E.coli. Fortunately, Staphylococcus aureus phages, which are a more urgently required form of therapy, has much lower resistance . This again shows that resistance to phage therapy is often detrimental to the bacteria itself. It may lose virulence towards the host or decrease resistance to other antibiotics . This has been shown in the case of multidrug-resistant P. aeruginosa, where the antibiotic sensitivity was restored . Despite this identification, this is not always the case, and in some cases, it is notable that it is of no detriment to the bacteria at all . There is no sign that anti-phage therapy is developing alongside antibiotic resistance .
Persisters in a population of susceptible bacteria can demonstrate tolerance to phages. This tolerance takes place without genetic mutation . When the phages are no longer present, they can revert back to their growing state . This tolerance is not passed down through generations. However, the time that it takes to allow the phages to pass, means that following this growth, there is enough time to re-establish a colony, within which, there may be the development of genetic resistance.
This would suggest that for the complete elimination of the bacterial population, treatment would need to be implemented in a periodic fashion that did not delay for a substantial length of time.
Decreasing resistance
It may be the case that a resistance proof phage cocktail could be produced if the range of targets is wide and the structures on the targets are conserved. Natural selection causes the development of systems such as CRISPR/Cas, rather than the production of resistant clones. An issue with potential resistance proof phage cocktails is the limited specificity to targets, meaning that a constant review system would be needed to maintain efficacy. Even when resistance is present, there is still a clear advantage to applying unmodified strain of phages with the relative decreased harm that phages will cause to the patient .
Using the method determined by Appelmans in 1921, there are ideal phages that can be utilised in order to optimise the treatment . There are ideal elements to cocktails too that can be implemented in order to reduce the resistance that may occur. Through different absorption factors, the levels of antiviral targeting of highly conserved regions of the bacteria can maintain a low level of resistance. This is due to a spread of natural selection. It is often suggested that phage therapy would work as a last line treatment alongside antibiotics, and this would be another way to negate the effect of resistance . This would also help to synergistically regain the efficacy of some of the antibiotics . There is also the option of using a series of different phages one after another in order to select for a wide range of resistant species . In acute infections, the time taken for the research into the infection and the pathogen responsible would require extremely expensive technology. This would be less practical than using a phage cocktail with a range of phages. There could be the option of using a burst release of the phages in order to satisfy a sequential approach while keeping costs low .
Phage Training
As described earlier, by exposing phages to an isolated bacterial population, the effectiveness of this phage against the ancestral species of the bacteria is increased. Bacterial populations are known to show heterogeneity that can arise from either genotypic diversity or phenotype differences within a genetically identical bacterial species . This causes an issue when trying to identify a treatment that could elicit an absolute antibiotic effect .
In an example of a personalised phage therapy case in 2017, the resistant clones that were in the patient’s body were chosen and isolated. This resulted in a necessitated change in the composition of the phage cocktail and acted as evidence for this heterogenicity . In order to overcome this, the training of the phages can level out the advantages of the bacterial target. Phage training techniques have been developed since the original method by Appelmans in 1921. There are two most commonly seen.
The first is a coincubation of bacteria and the phage which targets the bacteria. A filtration then takes place following treatment with chloroform . In the second method, the dilution of a mixture of the two organisms is placed in a growth medium for incubation . Both of these methods can be repeated using fresh ancestor bacteria until the most efficacious phages have been naturally selected.
Bacteria may lose virulence towards their host or decrease resistance to other antibiotics . This has been shown in the case of multidrug-resistant P. aeruginosa, where the antibiotic sensitivity was restored . Studies that took place in vivo, such as in murine models, showed that the infectivity of the phage is vastly improved through phage training. PAK_P3 went from only slightly lysing a Pseudomonas aeruginosa, to targeting it with 100% effectiveness in the lung . This was a result of an open reading frame change. Increased infectivity infers a decreased level of resistance present in the bacterial strain.
Although phage training has been common practice for almost a century, it is still clinically relevant today. It plays a valuable role in the implementation of the therapy and has more than one advantage. Not only does it allow for a broader target spectrum that shows high variability in terms of their phenotype and genotype; it also allows phage cocktails to be kept to a smaller variety of phages. However, a drawback becomes evident when considering the use of phages in intensive care settings.
There is a minimum time frame for the use of phage training, which is over 24 hours . In acute infections, patients can often require treatment in the space of minutes and, as such, phage training would prove of limited use. If the bacterial strain can be identified at an earlier point before the patient reaches a critical state, phage training can be implemented. If not, a wide-ranging phage cocktail would be a more suitable treatment. Despite the use of phage training for almost a century, there is limited data evaluating of the technique. To fully understand the cost and effectiveness of it, an increase in clinical trials is required.
As phages are naturally sourced and left to act as they would outside of trials and laboratories. Therefore, regulation for trained phages and monophages should be kept the same. While trained phages show variation from those their untouched counterparts, it is simply a quicker evolutionary process.