*2.4.2 Phage replication: Lytic and lysogenic cycles*

Frequently, *S. aureus* displays prophages inserted into its DNA and this viral genetic material contributes to bacterial adaptability once it encodes virulence and fitness factors [34]. Although most phages that infect *S. aureus* are temperate, i.e. lysogenic, some of them are strictly lytic and present potential for use as antistaphylococcal agents. According to the International Committee on Taxonomy of Viruses (ICTV), phages with DNA genetic material belong to the order *Caudovirales* which comprises nine different families: *Siphoviridae, Myoviridae, Podoviridae, Herelleviridae, Drexlerviridae, Demerecviridae, Chaseviridae, Autographiviridae* and *Ackermannviridae* [35]. The phages described so far capable of infecting *S. aureus* belong to the first three families, of which *Myoviridae* and *Podoviridae* involve *S. aureus* phages whose cycle is exclusively lytic [36]. Phages from these families are characterized by having an icosahedral capsid, where the genetic material is located, and are differentiated by the type of tail they have, which can be long and flexible (*Siphoviridae*), long and retractable (*Myoviridae*) or short (*Podoviridae*) [18].

Regardless of the type of cycle (lytic or lysogenic) performed by the bacteriophage, the replication process will begin by the adsorption of the virus to receptors on the surface of the host cell wall. During the infection of Gram-positive bacteria, as is the case of *Staphylococcus* spp., proteins present in the fibers of the viral tail interact with the teichoic acids of the cell wall, and the teichoic acids found in *S. aureus* are distinct from those observed in other *Staphylococcus,* thus allowing the specific binding of the phage [37]. The absence of this receptor in the bacteria renders the phage unable to bind and start its replication cycle, giving the virus the characteristic of being host specific. After the irreversible binding of the phage to the bacterial proteins, the bacterial cell wall undergoes the action of enzymes associated with the phage tail tip complex, forming a pore in the bacterial wall through which the genetic material of the virus is ejected into the cell. In *Staphylococcus* phages of the *Myoviridae* family the ejection of the viral DNA is facilitated by the contraction of the tail sheath [38]; in *S. aureus Siphoviridae* phages occurs the action of enzymes associated with the phage tail tip complex [39] and in *S. aureus Podoviridae* phages are the putative cell wall-degrading enzymes located in the tail spike [40]. Once the viral DNA is inside the host, either the lytic or the lysogenic cycle will be performed according to the characteristics of the phage.

The lysogenic cycle is characterized by phages that are able to infect and integrate their genetic material into the DNA of the bacteria, thus forming a prophage. The ability to integrate its genetic material with the bacteria is due to the presence of genes that encode the integrase protein, an enzyme that mediates the recombination between the phage's DNA and that of the host [41]. Subsequently, proteins are produced that induce viral latency, implying a pause in the transcription of gene products, allowing the prophage to exist with the bacteria for several bacterial

generations without major consequences. Furthermore, the prophage induces immunity in the bacteria against infection via new phages. Bacteriophages that exhibit this type of replication cycle are not suitable in the context of phage therapy, since at the end of the viral cycle the death of the bacteria will not necessarily occur. In addition, bacteriophages that perform the lysogenic cycle may be responsible for producing toxic substances and carrying resistance genes [32], implying benefits for the bacteria.

On the other hand, in the lytic cycle there is no integration of the phage genetic material to the prokaryote DNA. At the end of this viral replication cycle, when the new virions are already formed and ready to be released, there is the production of enzymes capable of lysing the bacteria cell wall, inducing bacterial rupture and death for the release of new virions. Therefore, phages whose replication cycle is lytic are the most suitable for use in phage therapy, precisely because they cause bacterial lysis [18]. The schematic representation of the lytic and lysogenic cycles in *S. aureus* is shown below (e.g., **Figure 1**).

#### *2.4.3 History of phage therapy in S. aureus infections*

The attempt to use phages for the treatment of infections caused by *S. aureus* began soon after the discovery of phage therapy, and it is likely that the first use was in six patients with skin diseases in 1921. After the discovery of antibiotics, the studies related to phage therapy were abandoned and the few that continued, conducted in Georgia, Russia, and Poland, included efforts to treat staphylococcal infections [31]. Although the main studies target the use of phage therapy in humans, phages have also been proposed for use in veterinary medicine. The first case of application of this therapy in animals was associated with d'Herelle, one of those responsible for the discovery of phages. In 1919, he used the viruses to contain an outbreak of lethal typhoid fever in chickens. After analyzing several dead animals, d'Herelle was able to identify *Salmonella* Gallinarum and after isolated a lytic bacteriophage for the bacterium in question [42]. In another study, *S. aureus* phages were tested in mice, but the results were unsatisfactory because the virus used was not able to protect against a lethal dose of the bacteria [42].

Studies with phages for the control of staphylococcal infections were continued in some regions of the world. In the United States (1952), a laboratory (Delmont Laboratories) licensed, for human use, a bacterial lysate produced from the

**Figure 1.** *Lytic and lysogenic cycles.*

#### *Bacteriophages as Anti-Methicillin Resistant* Staphylococcus aureus *Agents DOI: http://dx.doi.org/10.5772/intechopen.98313*

infection of bacteriophages in two virulent strains of *S. aureus.* Several years later, in 1986, the same product was licensed for veterinary use for the treatment of recurrent canine pyoderma but is no longer marketed for human use. This lysate, whose commercial name is "Staphage Lysate SPL", consists of bacterial cell wall fragments, intracellular components released during bacterial lysis, culture media ingredients, and viable bacteriophages. In 1981, it was demonstrated to be able to protect 80–100% of infected mice compared to the group not treated with SPL [43]. In dogs, SPL has been used effectively to treat chronic staphylococcal blepharitis as well, where weekly injections were administered to control the disease without adverse effects on the animals [44].

Because of the resistance of *S. aureus* to antimicrobials, some studies have sought to evaluate the activity of phages and their products against MRSA isolates. In 2008, one study evaluated the potential use of phages to eliminate or reduce nasal colonization by *S. aureus*, concluding that decolonization may be beneficial for certain patient groups, and phages were able to effectively combat induced infections in animal experiments [45]. A recent review concluded that phages are effective as topical antimicrobials against *S. aureus*, being able to combat MRSA in skin infections regardless of whether they are used with or without combination to topical antibiotics [46]. In addition to the phage itself being used as an antimicrobial agent, its products, such as lytic enzymes (endolysins), are also the subject of investigation. Phages and their products can be administered orally, inhaled, intravenously, subcutaneously, and topically, as suspensions for ocular use or application to bacteria-infected burns. The use of bacteriophages in therapeutics has advantages, mainly the high viral specificity that allows them to bind only to bacterial cells with the specific receptors, not affecting human or animal cells, thus avoiding significant side effects. Furthermore, phages can be used in the control of bacteria that show resistance to antibiotics [32]. Additionally, these viruses can adapt to the resistance mechanisms developed by bacteria, evolving in parallel to their host.

### *2.4.4 Commercial phage products anti-staphylococcal*

Commercial products containing phages or enzymes produced by them are manufactured and available in some countries, mainly in Russia and Georgia, but also in Canada, the Netherlands and the Czech Republic. The following table (e.g., **Table 1**) gathers different commercial phage products, the target bacteria of each product, their main uses and the manufacturer [47–50].

In recent years, different studies involving commercial phage products with anti-staphylococcal activity have been undertaken. Most of them were related to *S. aureus Myoviridae* phages and demonstrated very promising results. Among them, it was shown that 100% (10/10) of multidrug resistant *S. aureus* isolates were lysed by Fersisi phage cocktail; 90% (9/10) were lysed by Instesti bacteriophage and 80% (8/10) by Pyo phage cocktail, showcasing the high lytic activity of commercial phage cocktails of Eliava BioPreparations, Georgia [51]. Similarly, 95% of clinical isolates of staphylococci, including 3 MRSA and 17 Methicillin susceptible *S. aureus* (MSSA) were sensitive to the action of Pyofag® polyvalent bacteriophage (Pharmex Group LLC, Ukraine for NeoProbioCare Inc.) Moreover, the same commercial phage cocktail was able to control furuncles in a patient with skin lesions by topical application of Pyofag®, as well as orally and nasally, for 14 days [52].

Some commercial products with the same name, but produced by different manufacturers, are proposed for the control of *S. aureus* in skin and wound infections, including Pyophage (polyvalent purified) cocktails from Microgen (Russia) and Pyophage from Eliava BioPreparations (Georgia). One study evaluated the performance of both cocktails against 20 MSSA and 31 MRSA clinical isolates


*Bacteriophages as Anti-Methicillin Resistant* Staphylococcus aureus *Agents DOI: http://dx.doi.org/10.5772/intechopen.98313*


#### **Table 1.**

*Commercially available anti-S. aureus phage products.*

and concluded that both products had greater than 75% coverage, but Microgen's Pyophage was extremely effective against MRSA, killing 97% of the bacterial isolates. Genomic analyses of the *S. aureus* phages contained in these commercial products revealed great similarities (*Myoviridae, Kyavirus* genus), however Microgen's cocktail additionally featured a *S. aureus Podoviridae* component that possibly contributed to the higher coverage observed against MRSA [53].

In a recent study, the action of Stafal® (a preparation with polyvalent bacteriophages active *on S. aureus)* on planktonic cells as well as on biofilms produced by MSSA and MRSA was demonstrated. Bacterial cells immersed in the biofilm required high phage concentrations and longer exposure time to be destroyed compared to planktonic forms [54]. It is likely that this occurred because of the difficulty of the phage to access the host cell surface within the biofilm matrix. Still, the phages were active on the biofilms, whereas antimicrobials are known to be ineffective due to the limitation of their diffusion through the extracellular polymeric substances matrix. Similarly, enzymes encoded by bacteriophages called endolysins have shown promising advances against bacterial biofilm formation. Such enzymes are responsible for lysis of the host bacterial cell wall promoting the release of viral progeny at the end of the replication cycle of lytic phages [55]. Experimental assays showed that the phage-derived lysine named "LysH5" was able to remove *S. aureus* biofilm, even eliminating persistent cells (subpopulation of cells that showed high resistance to antibiotics). During treatment of staphylococcal biofilm with LysH5 (0.15 μM), complete inhibition in biofilm formation was also seen in certain *S. aureus* isolates [56].

Commercially, the recombinant endolysins Staphefekt SA.100 and Staphefekt XDR.300 (Micreos Human Health BV, Netherlands) which act on *S. aureus* (including MRSA) are available for use. A few clinical studies have been conducted with Staphefekt SA.100 and all have demonstrated remission and/or improvement of chronic *S. aureus* skin infections (folliculitis, rosacea, and eczema) [57, 58], reinforcing the utility of this therapeutic resource. Moreover, it is believed that endolysins may be better therapeutic alternatives than bacteriophages themselves since bacteria have the possibility to develop resistance to the phage. On the other

hand, it is necessary to consider that endolysins present limitations, such as: i) induction of inflammatory response of cytokines and neutralizing antibodies that imply the reduction of the half-life time (*in vivo*); ii) their systematic use *in vivo* will provoke an immune response that will promote the loss of the lytic activity of the enzyme [59]; iii) lower activity on Gram-negative bacteria due to the presence of the external membrane in the cell wall [60].

Other commercially available products are: Bronchophage, Otophage, Phagodent, Phagoderm, Phagogyn, Phagovet, Vetagyn (Micromir, Russia); ENKO bacteriophage, SES Bacteriophage, Staphylococcal bacteriophage (Eliava BioPreparations, Georgia); Dysentery bacteriophage, *E. coli* bacteriophage, *E. coli-Proteus* bacteriophage, *Klebsiella* purified polyvalent bacteriophage, Sextaphag® polyvalent pyo bacteriophage, *Streptococcus* bacteriophage (Microgen, Russia); Phagestaph, Phagyo, Septaphage (Biochimpharm, Georgia); and Intestifag® polyvalent bacteriophage (Pharmex Group LLC, Ukraine for NeoProbioCare Inc., Canada). Detailed information can be found in related sources [47–50].

#### *2.4.5 Non-commercial anti-S. aureus bacteriophages*

Fortunately, since the year 2000, different studies have contributed to a better understanding of phages as anti-*S. aureus* therapeutic agents. For example, the efficacy of the bacteriophage named ØMR11 against a lethal infection caused by *S. aureus* in mice was evaluated. Initially, the phage was isolated, had its bacteriolytic activity determined, and finally, *in vivo* infection experiments were performed by introducing *S. aureus* intraperitoneally, including MRSA strains, causing bacteremia and eventual death of the mice. After peritoneal administration of the isolated phage in infected animals, suppression of *S. aureus*-induced lethality occurred [61]. Similarly, the use of cloned lysins encoded by the phage ØMR11 was efficient in cell lysis, including MRSA. These lysins are enzymes produced at the end of the replication cycle of bacteriophages and are responsible for degrading the bacterial wall and releasing virions. After sequencing the phage ØMR11 the possible genes related to lysins were identified, these were cloned, and their protein products were purified on a large scale. The results showed high activity of lysins against MRSA isolates both in mice contaminated intranasally and subsequently treated with the intranasal lysins, as well in animals infected intraperitoneally, showing that the enzyme can be used for the control of *S. aureus* in humans and domestic animals [62].

A cocktail containing two bacteriophages, designated K and 44AHJD, was tested against clinical isolates of *S. aureus*, showing 85% of lytic action on the bacteria. The *in vivo* efficacy of the cocktail was evaluated through the murine nasal colonization model. Efficient decolonization was verified after eight days of intranasal administration in animals treated with the phage cocktail, while the control group (received only the bacteria) and the group treated with placebo remained colonized [63]. Although different studies have already demonstrated the efficiency of phages on *S. aureus*, few clinical trials have been conducted to validate their efficacy and safety. According to the records of clinical trials involving *S. aureus* and bacteriophages, in progress or already concluded [64] it appears that they are scarce and that few countries, mainly the U.S., have invested in clinical trials that corroborate the use of phages in clinical practice (e.g., **Figure 2**). The lack of large clinical studies that can effectively consolidate the use of phages *in vivo* is an obstacle to be overcome.

The Clinical Trials platform, a database of clinical studies conducted worldwide, reports the existence of eight studies related to the use of bacteriophages against *S. aureus* [64]. These are intended for the use of viruses for the treatment of ulcers infected by *S. aureus* in diabetic patients, prevention, and treatment of infection by *S. aureus* and other bacteria in burn patients, use for patients with covid-19

### *Bacteriophages as Anti-Methicillin Resistant* Staphylococcus aureus *Agents DOI: http://dx.doi.org/10.5772/intechopen.98313*

#### **Figure 2.**

*Clinical trials involving* S. aureus *and bacteriophages. Available at: www.pngwing.com*

affected with pneumonia or bacteremia/septicemia due *S. aureus* infection, use in patients with serious or immediate risk of life, and patients with venous leg ulcers. In addition, three studies that use phages as a diagnostic method. When considering regulatory measures for the application of phages as therapeutic agents, it is likely that, initially, such viruses are more easily used prophylactically in order to reduce the frequency of infections. In contrast, phage therapy aimed to eradicate systemic bacterial infections will inevitably be more complex [65].
