Phage Therapy: A Potential Novel Therapeutic Treatment of Methicillin-Resistant Staphylococcus aureus

The emergence of multidrug-resistant bacterial strains, especially in the clinical setting, has renewed interest in alternative treatment methods. The utilization of prokaryotic viruses in phage therapy has demonstrated potential as a novel treatment method against multidrug-resistant bacterial infections. As the post-antibiotic era quickly approaches, the development and standardization of phage therapy is critically relevant to public health. This review serves to highlight the development of phage therapy against methicillin-resistant Staphylococcus aureus (MRSA), an antibiotic-resistant bacterial strain responsible for severe clinical infections.

mortality rate of MRSA infections is approximately 32 percent 21,22 . Considering this, the development of alternative treatment methods is essential to the preservation of public health. Figure 1 depicts the chronological map of S. aureus treatment. In 1940, the discovery of penicillin as a miracle drug offered unlimited hope to bacterial control; however, within the space of two years, S. aureus developed resistance to penicillin 7,[23][24][25] . By 1960 over 80% of S. aureus strains had developed resistance to penicillin 23,25 . Methicillin was introduced in 1961 as an alternative treatment of S. aureus.
Only a year later, S. aureus developed resistance to this antibiotic as well 25 . The first outbreak of MRSA was recorded in 1968, followed by the second and third outbreaks between 1970 and 1980 25 . By 1980 MRSA had spread worldwide.
In 1990, vancomycin became the drug of choice against MRSA 25,26 . However, there was an observed rise in intermediate vancomycin resistance, leading to the occurrence of complete vancomycin resistance in 2002 25,26 . Since 2002, MRSA prevalence and a decrease in antibiotic development created a severe risk to public health. Several researchers have delved into antibiotics against MRSA; however, none have reached clinical applicability 27,28 . For instance, in 2019, Nicolas et al. showed that peptidomimetics-cyclic heptapseudopeptides were effective against MRSA in both mild and severe sepsis, and these antibiotics did not pose any health threat to humans in an in vitro study 27 . Further animal studies also confirmed that there was no toxicity recorded for mouse models and zebrafish embryos 27 . In another study in the same year, Geitani et al. reported that two novel peptides named "LL-37 and CAMA" were potent against clinical isolates of MRSA 28 . The progress of antibiotic development for MRSA has since declined due to the cost of production for these highly specialized semi-synthetic compounds. Due to this decline in antibiotic drug development paired with the increased cost of these drugs, bacteriophage therapy has once again become relevant in the field of therapeutics. In 2009, a group of researchers examined the safety of bacteriophagebased formulations for treating wounds caused by S. aureus 29 . In phase I clinical trial, they reported no safety concerns with the use of bacteriophage treatment; nonetheless, they encouraged a vigorous test for the efficacy of the phage preparations in a phase II trial 29 . In 2013, a bacteriophage lysin named "PlySs2", an aminopeptidase was reported to have bactericidal activity, exhibiting a MIC of 16 μg/ml for MRSA with a single dose of 2-mg of PlySs2 being potent enough to confer 92% protection against MRSA in mice 30 . This peptide showed notable broad lytic activity also against S. pyogenes, with high thermostability, hence presenting as a good candidate for MRSA therapeutic 30 .

Virulence Factors Associated With MRSA
Antibiotic resistance is the primary clinical obstacle for the treatment of MRSA infections. Therefore, it is crucial to understand the virulence factors that facilitate this resistance. MRSA infections are resistant to beta-lactam antibiotics such as penicillin and semi-synthetic antibiotics such as methicillin, which were the standard treatment of S. aureus before MRSA 23 . To understand the virulence factors that allow for MRSA's antibiotic resistance, it is essential to also understand the evolution of S. aureus infections. As figure 1 outlines, S. aureus has gradually developed resistance to antibiotics, starting with penicillin in the form of penicillin-resistant S. aureus (PRSA), which was first reported in 1942 7,24 . The virulence factor present in PRSA was determined to be the gene blaZ 7,31 . This gene inhibits the binding of penicillin-binding proteins (PBPs) that function to disrupt peptidoglycan cross-linking during cell wall synthesis 23 .   As shown, the dimeric PBP 2a binds to peptidoglycan moiety from MRSA. The PBP 2a structure (3ZG5) was extracted from the RCSB website (https://www.rcsb.org/structure/3ZG5), with the PBD ID, 3ZG5 35 . Molecule -ligand interactions were analyzed using Biovia Discovery Studio 2021 Client (BIOVIA Discovery Studio Visualizerhttps://discover.3ds.com/discovery-studio-visualizer). As shown in figure 2A, the dimeric molecule binds to peptidoglycan via a minor cleave found in both monomers (chain A, and chain B

C B A
lactam acylation; hence the dd-transpeptidation reaction is carried out, thus producing the cell wall of the bacteria.
As shown in Figure 3, the PBP 2a enzyme, a dimeric molecule, is shown bound to a peptidoglycan moiety during the bacterial cell wall synthesis process.
The ability of MRSA to acquire mobile genetic elements carrying a variety of virulence factors has led to significant variation among MRSA strains 24 . Virulence factors that have been highlighted in literature include Panton-Valentine leucocidin 24,34 (PVL), PSM cytolysins, and toxic shock syndrome toxin-1 24,35 . These exotoxins are responsible for MRSA's increased virulence and exceptional ability to evade the immune system 24 . The evolution of S. aureus and its virulence factors has increased the threat of these infections to public health. A list of some of the virulence factors of MRSA is shown in Table 1.

Virulent Factor
Function Reference

S. aureus Biofilm as a Physical Shield Against Antibiotics
The formation of biofilms by Staphylococcus spp is a crucial adaptation for bacterial survival, thus protecting it from harsh environmental factors, antibiotics, and even the bacterial host immunity 42 . In Staphylococcus epidermidis, the discovery of poly-N-acetylglucosamine (PNAG) and polysaccharide intercellular adhesin (PIA) was the first factor shown to mediate biofilm formation 43,44 . The discovery of multiple biofilm formation factors in S. aureus such as the LPXTG-cell wall-anchored biofilm-associated protein (BAP) 45 , fibronectin-binding protein (FnBP) 46

Lytic Phage for Phage Therapy
Phages suggested for phage therapy utilize a lytic mechanism for the infection of bacterial cells. The lytic lifestyle is comprised of five stages: attachment, penetration, biosynthesis, maturation, and lysis. In the attachment stage, phages utilize their tailspike proteins to interact with specific bacterial surface receptors of the lipopolysaccharide membrane. This interaction has been observed at the molecular level in a variety of phage families [50][51][52] . As previously mentioned, phages are characterized by a narrow host range and may infect only one species or strain of bacteria within a species 53 . This specificity is unique and can be exploited for targeted treatment of bacterial infections in phage therapy and identification of bacterial pathogens in phage typing 54,55 . Following attachment to the host cell membrane, the phage utilizes its tail machinery to penetrate the cell membrane and inject its viral genome 56,57 . aureus phages that carry out lytic lifestyles in MRSA is a vital step in the development of viable treatments. Isolation of phages from the order Caudovirales followed by characterization and in vitro testing is a viable method for identification of S. aureus phages with lytic lifestyles within MRSA [66][67][68][69][70] . Phages are known to be abundant in any ecosystem in which their bacterial host is present 68 . Literature has been able to utilize samples, primarily from healthcare facility sewage, for the isolation of S. aureus phages [66][67][68][69] . Characterizations of these isolates through doublelayer plaque assay (DLA) and electron-microscopy have resulted in the identification of S. aureus phages belonging to the Siphoviridae and Myoviridae families [66][67][68] .
Phages of the order Caudovirales are classified structurally into three families of tailed bacterial viruses: Myoviridae (long contractile tails), Siphoviridae (long non-contractile tails), and Podoviridae (short non-contractile tails) 67 . One of the renowned prototypic phages from the Podoviridae is the Salmonella phage known as P22 71 and its phylogenic relative the ɛ34 phage, which also infects Salmonella spp 72    showing promise for this method 60 .

Host Range
While lytic phages are considered the standard for phage therapy, there are still some concerns about their abilities. Scientific understanding of phages has been greatly advanced since their discovery a century ago. However, our knowledge of phages is still limited. The genomes of lytic phages can contain greater than 50% hypothetical genes with no known function 86 , as well as encode auxiliary proteins that alter bacterial physiology in ways that are not fully known 86 . The number of genes and auxiliary proteins that we are currently unaware of makes abortive infections a major concern. Abortive infection is a method of bacterial defense in which the bacterial cell upon infection kills itself to ensure the replication of a phage is stopped. This mechanism could possibly lead to the bacterial host acting as a reservoir inside the human body for phage DNA with unknown functions. This concern is also shared with mutant phages such as vir and clear plaque, especially considering that temperate phages typically carry a wide range of virulence factors 60 . Continued research of phage genetics is key in ensuring the safety of phage therapy.

Comparison of phages to Antibiotics
Phages and antibiotics both serve as antibacterial agents functioning to lyse or inhibit the persistence of bacterial infections. While both agents have a similar function, they feature several key differences that determine their appropriateness for situational usage.
The use of antibiotics has been observed to have adverse health effects in some situations 87 . Adverse health effect of antibiotics includes instances of anaphylaxis, nephrotoxicity, cardiotoxicity, hepatotoxicity, neurotoxicity, and several gastrointestinal and hematological complications 87 . The most common adverse effect of antibiotic treatment is an allergic reaction, which is prominent in children 87 . These allergic reactions are most commonly the product of high tissue concentrations [88][89][90] . The safety of phage therapy has not been as extensively studied, especially in western medicine. However, new studies have deemed phage therapy practices such as oral administration as safe [89][90][91][92][93][94] . In oral administration, the translocation of phage across the intestinal epithelium into the blood has been suggested as beneficial to the host 95 . The benefit of this translocation is the downregulation of immune response to indigenous gut microbiota antigens through the inhibition of interleukin-2, tumor necrosis factor, and interferongamma production 95 . This downregulation, in addition to phage host specificity, protects the natural gut microbiota.
The protection of natural gut microbes is a typical criticism of antibiotics. The immunological response to phage therapy may be beneficial in healthy patients; however, literature disputes the safety of treatment in patients with compromised immune systems [96][97][98] . The immunological response is especially significant in the context of MRSA infections that are prominent in patients who are immunocompromised. Patient-to-patient variation in the study of phage therapy has been an area of concern. While transduction may be beneficial to natural gut microbes, there is concern that this characteristic could also be related to the disruption of normal intestinal barrier function. This disruption could potentially lead to disorders such as Crohn's disease, inflammatory bowel disease (IBS), and type 1 diabetes 99 . Literature has determined that there is variation in the inflammatory response to phage therapy based on the site of infection 100 . The study of phage therapy is relatively new, and there are many characteristics such as immunological response and physiological response that require further study to comprehensively assess the safety of phage therapy.
Host specificity is a defining characteristic of phage therapy. The broad use of antibiotics has been documented for their adverse effects on the human gut microbiome that sometimes lead to diarrhea and C. difficile infection 101 . Other potential outcomes of antibiotic perturbations in the gut microbiome include asthma, obesity, and diabetes [102][103][104] . Phage therapy is highly specific to bacterial species and strain, resulting in less irritation of the natural gut microbes while still effectively reducing the presence of pathogens 105,106 . As discussed in the host range section of this review, the specificity of phages can sometimes lead to the inability to treat an infection colonized by multiple bacterial species. A common clinical example of this scenario is burned victims who typically suffer infections colonized by more than one singular bacterial strain 107  large-scale production, and distribution, a distinct advantage of broad-spectrum antibiotics.
An interesting characteristic of phage therapy is the relationship between geographic location and phages used for treatment. Studies have shown that phages show high specificity to bacterial targets from their indigenous region 94,108 . These studies utilized Russian E. coli phage cocktails for the treatment of microbiologically determined E.
coli diarrhea in Bangladesh 94 . The treatment resulted in no improvement of clinical outcome. Results suggested that phage cocktails are better adapted to local bacteria populations 108 , and that bacterial host range can be restricted both spatially and temporally 109 . A suggested solution to this challenge is the development of phage cocktails with regional specificity for the clinical setting 110 . In the context of MRSA infections, as well as other antibiotic-resistant bacterial strains, this means that the phages that can be used to target these bacteria are likely found in the same enviroment 111 .
While this high specificity provides challenges in production that are not common with broad-spectrum antibiotics, it does have some benefits. Regions that have limited access to antibiotics would greatly benefit from the ability to isolate phages that could be utilized for specific phage therapy of regionally prevalent pathogens. The utilization of phage therapy in these regions would also positively impact the economic burden that the cost of antibiotic treatment entails. Antibiotics have been a cornerstone of clinical treatment for over a century, but the increased prevalence of antibiotic-resistant bacterial strains has required the development of new novel treatments. The limited adverse effect, target specificity, and abundance of phages in the natural world make phage therapy a potentially viable treatment.   Table 2. Mechanisms of Therapeutics against S. aureus A brief comparative description of antibiotic and potential phage S. aureus treatment methods, mechanism, and resistance.

Clinical Challenges of Phage Therapy against MRSA
The lack of validated and adequately controlled clinical trials is a challenge to progressing phage therapy into standard clinical practice 113 . The pharmacological characteristics of phages hinder their standardization in clinical trials. A primary pharmacological concern is the self-replicating nature of phages; unlike conventional drug treatments, phage therapy requires awareness of various novel kinetic phenomena 114  While phage monotherapy has shown promise, combination therapy or phage cocktails also offer a broad range of activities against bacterial infections. Phage cocktails, as previously described in this review, consist of the combination of several phages with various host ranges. This combination addresses the limitations of monotherapies host range and reduces the potential development of phage resistance in bacteria. While phage cocktails feature a broader host range, it has been shown that they significantly increase the challenge of assessing inflammatory response, potential gene transfer, and the development of multi-phage resistance 117 . Further study and standardization of phage cocktail therapy are required to determine their effectiveness as well as efficacy fully.

Human Clinical Trials
Human clinical trials for phage therapy against MRSA are limited due to the challenges previously mentioned.
Standardization of clinical trials requires preliminary studies to determine the adequate dosage, delivery, and host response. The use of animal models has mainly been beneficial to the progression of standardized phage therapy  Table 3. Strains of S. aureus, the antibiotic they developed resistance against, and the potential phage treatments options.

Conclusion
The increased prevalence and occurrence of antibiotic-resistant bacteria is a major threat to public health, especially the notorious antibiotic-resistant S. aureus. While antibiotic dose-response has been standardized, consideration of MRSA phages varied replication factors is crucial for the determination of standard relative dosage for 'killing' titers. Additionally, MRSA phages multiplication is incumbent on host availability; for this reason, an initial "killing titer" might tremendously increase after phage administration through the phage's replicative process.
An added dimension in phage biology is its ability to co-evolve with its host; this added advantage over antibiotics enhances the need to study MRSA phages as therapeutic tools against the bacteria. Hence, a clearer insight into MRSA phage biology, pharmacokinetics, and pharmacodynamics will provide the requisite avenue for the broad application of phage therapy. It is undoubtedly that an alternative treatment method for these antibiotic-resistant bacteria such as MRSA is essential to counteract human infections 5 and the economic burden they present 12,13 . MRSA being one of the most prevalent antibiotic-resistant bacterial strains, is an immediate and severe threat to public health 5,20 . The utilization of lytic S. aureus phages for MRSA treatment shows potential as a treatment method. Literature has outlined the potential benefits of phage therapy against MRSA due to their host specificity, wide diversity, and success in animal and limited clinical trials. While phage therapy against MRSA requires further study, literature to this date suggests that phage therapy shows favorable potential as a novel treatment.