**4. Evolution of phage therapy**

between the N-acetyl-muramic in the glycan chain and the L-alanil residues; (2) endo-β-Nacetylglucosaminidases, which hydrolyzes the N-acetylglucosamine β-1,4-N-acetylmuramine acid linkage; (3) N-acetyl-muramidases, which catalyze the hydrolysis of N-acetylmuramoilβ-1,4-N-acetilglucosamine bond; (4) transglycosylases, which disrupt β-1-4 glycosidic bonds by forming a 1–6 anhydride ring in the N-acetylmuramic residue; (5) endopeptidases, which may hydrolyze both the tetrapeptide linked to the glycosil moieties and the pentapeptide

Endolysins encoded by double-stranded DNA bacteriophages have a molecular weight between 25 and 40 kDa [33]. Most of endolysins are composed of at least two functional domains: one containing the catalytic activity located generally in the N-terminal domain and one responsible for the recognition of a specific substrate associated with the C-terminal domain. In some cases, more than one catalytic domain or more than one recognition domain are present [19]. The recognition domain usually joins to specific molecules in the bacterial cell envelopes such as monosaccharides, coline or teichoic acids [34]. Endolysin activity is usually species specific, although there have been reports of endolysins with a wider substrate range. Besides, the cell wall recognition domain is not always essential for endolysin activity. The endolysin got a wider substrate range, but it conserved certain specificity, since it was no active against all bacteria. Studies of crystallography and mutation analysis with endolysin *PlyL* against *Bacillus anthracis* led to propose that the C-terminal domain of this endolysins inhibits the activity of the catalytic domain by particular intermolecular interactions. This inhibition is released when the C-terminal domain binds to its particular ligands in the target cell wall, thus acting as a regulatory domain [35]. Most of the reported endolysins from phages against *S. aureus* have two catalytic domains and a cell wall recognition domain being LysK one of the must studied endolysin models. LysK has a cysteine/histidine-dependent aminohydrolase/peptidase (CHAP) catalytic domain that hydrolyzes the peptidic bond between the D-alanine of the oligopeptide chain attached to the sugar backbone and the first glycine of the pentaglycine bridge that is typical of *S. aureus* peptidoglycan and confers resistance to lysozyme. CHAP presents the higher activity of both hydrolytic domains. LysK also has an N-acetylmuramoyl L-alanine amidase or amidase-2 (Ami-2) catalytic domain which catalyzes the hydrolysis of the N-glycosidic bond between the N-acetylmuramic residue and the L-alanine of the oligopeptide attached to the sugar backbone. A third domain called SH3b is responsible for the specific recognition of cell wall components, strain specificity and modulator of hydrolytic activities [36, 37]. Endolysin 2638A has similar triple domain structures: an amino-terminal domain with endopeptidase activity, a central Ami-2 domain (with the highest activity in this phage) and a SH3b cell wall recognition domain [38]. Modular structure of *S. aureus* endolysins has allowed the construction of chimeric endolysins by the combination of catalytic and/or recognition domains. An example is the endolysin Ply187AN-KSH3b, which is a translational fusion of the CHAP domain of phage Ply187 and the cell wall recognition domain SH3b from LysK endolysin. This endolysin was effective in a mouse model of endophthalmitis that also decreased inflammatory response and protected the retina from tisular

entrecrossing bridge [31, 32].

186 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

damage [39].

Since the discovery of bacteriophages, it raised the idea of using them for treatment of bacterial infections. D'Herelle began testing the therapeutic effects of phages, using animal models such as chickens and cows first, which provided successful results. Subsequently, there was carried out human testing and the development of phage therapies became more extensive. In 1923, the development of phage-based therapy strengthened with the foundation of the Eliava Institute in Tbilisi, Georgia, in the former USSR. In 1940, they began to commercialize phage in the United States. During World War II, phage cocktails were used to treat diseases such as dysentery and gangrene in the soldiers of the former Soviet Union. Their application was topical, oral and intravenous, although the latter favors the immune response of the individual treated due to the protein content of the virus, resulting in the elimination of the phage from the body [40–42]. Until a few years ago, therapies were based solely on the administration of the complete bacteriophage, but it was until 2000 that the studies for the identification and purification of lytic enzymes to treat infections caused by bacteria begun. In addition to using bacteriophages and their enzymes as enzybiotics (enzymatic activities with antibiotic effect) in the treatment of infections in humans, animals and agriculture, they are also used in the food industry as preservatives and disinfectants [19]. After the discovery of penicillin, the development and commercialization of antibiotics in the 1940s and 1950s soon occupied the global antibacterial market. The lack of knowledge of the biology of phages, the lack of studies of epidemiology of diseases and also a lack of control during the preparation of therapeutic


**Table 2.** Advantages of bacteriophages over antibiotics.

stocks led to a temporary delay in the research and development of phage therapy. In early studies of phage preparations, successful results showed high antimicrobial activity in *in vitro* and *in vivo* assays; however, in subsequent trials, some phages had little or no ability to destroy bacteria or became lysogenic [43].

Because of the concern in the treatment of diseases caused by pathogens with multiple resistance to antibiotics, it has revived the interest in the development and use of the bacteriophage therapy and their enzymes to treat diseases in animals and humans. Phage therapy has been used in plants, animals and humans with varying degrees of effectiveness; in addition, bacteriophages have some potential advantages over antibiotics but also have some disadvantages [44] (**Table 2**). The specificity of phage-host interaction permits the use of some phages in therapy because they do not have influence on normal microbiota in humans, animals, plants, food or inert surfaces. On the contrary, the use of broad spectrum antimicrobials has an effect on the eradication of a wide range of infecting pathogens but also kills bacteria from the natural microbiota thus causing a disequilibrium in the host normal microbiota and promotes secondary bacterial or fungi infections or even physiological or endocrinological disorders.

#### **5. Bacteriophages and its interaction with animals**

There is a high diversity of phages in microbial communities living in symbiosis with animals, for example, in the pig digestive tract and in the cow rumen [45, 46]. In the animals gut microbiota, there is a complex ecosystem with approximately 500 species of microorganisms, which are interacting with mutual benefits [47]. When the abundance of one of those bacteria changes and alters the dynamic equilibrium, it results in some disorders or disease in the host. Phages play an important ecological role for the health regulating the relative amount of the different bacterial strains in microbiota. On the other hand, the presence of phages in animals could present some disadvantages for health. When phages insert into the bacterial genome genes that encode toxins like Panton-Valentine, Shiga and diphtheria toxins [48, 49] or some other virulence factors, further excision may be aberrant, leading the phage genome to carry those virulence genes by transduction. These aberrant phages may insert in new hosts and transfer virulence properties. In fact, some genetic elements related to virulence may be originated from aberrant prophages. Also, prophages confer its host resistance to the infection of other phages. In addition, phages can also impact in host immune response through modifications in bacteria's antigenicity. Density of host bacteria determines the ability of phages to infect and reproduce because phages encounter their host through random collision. There are four models in the literature explaining the behavior of phages and bacteria in the regulation of animal microbiota. (A) "**kill the winner**": phages are more abundant than bacteria but don't infect them because of the lower abundance of its host, when some strains overgrow, phages can depredate and kill them by lysis, and system comes back to an initial healthy equilibrium. (B) "**kill the relative**": some phages are reproduced from lysogenic strains so they don't need to be abundant; strains with prophages produce phages that kill their genetically related strains which aren't resistant to the phage. The result is an advantage in the abundance of lysogenic population in comparison with non-lysogenic strains. (C) "**community shuffling**": temperate phages act negatively on their host, temperate phages kill their host under some stress situation and this don't occur with non-lysogenic strains. Positive feedback could take place if massive lysis causes host reactions like inflammation on another immune response. This causes an imbalance in the microbiota and in some cases disorders or diseases related to the change in populations. (D) "**invade the relative**": prophage propagates itself by infecting new hosts without lysing them, but establishing lysogeny [47]. Other contribution of phages to bacteria strains in animal microbiota is when phages function as vectors of virulence, for example, changing the expression of antigens in outer membrane like O-antigens [50], giving to bacteria genetic adaptation; it results in new and more virulent strains for the animal host.
