**7. Mastitis and** *S. aureus*

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

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 endocri-

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

bacteria or became lysogenic [43].

188 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

nological disorders.

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

Mastitis is characterized by the inflammation of the mammary gland in one or more quarters of the udder accompanied of leukocyte production, mainly monocytes and blood serum proteins such as cytokines, chemokines and interleukins [51]. It is caused mostly by contagious pathogens such as *S. aureus* and *Streptococcus* spp. and environmental pathogens such as *E. coli*. Also, in less proportion, mastitis can be caused by or promoted by injury, allergies and neoplasias [52]. Mastitis causes large economic losses in the milk and dairy products industry for about 2 billion of dollars each year in the USA [53]. Among the pathogens causing mastitis, *Staphylococcus aureus* is considered a causal agent of great concern because of the low cure rate of *S. aureus* infections by antibiotic treatment and its ability to persist in a herd in the form of undetected subclinical infections [54]. Vaccines for the treatment of mastitis have limited efficacy. Cure rates for antibiotic treatment are often lower than 15%. This is caused by the poor penetration of the gland by antibiotics allowing *S. aureus* to survive inside the epithelial or phagocytic cells. Antibiotic resistance in *S. aureus*is also a growing concern, with overall rates of antimicrobial resistance in bovine *S. aureus* isolates varying widely by region [55]. The continued emergence of MRSA strains in humans and animals points to the need to develop new antimicrobial agents or therapies treatment for this pathogen. The treatment of bacterial infections with bacteriophages and their derivatives is such an option. **Table 3** describes those approaches.


from streptococcal endolysin SA2 with either lysostaphin or LysK endolysin and the recognition domain of endolysin LysK

LysK-SH3B reduce *S. aureus* CFUs by 1–3 log units in cow milk and by 0.63–0.8 log units in mammary glands. Synergism with lysostaphin reduced CFUs by 3.36 log units


**Table 3.** Bacteriophages and endolysins therapy for treatment of *S. aureus* mastitis.

**Experiment Observations/treatment**

postinfusion

antibodies

Treatment consisted of 10 ml intramammary infusions of 1.25 × 1011 PFU of phage K and infusions with saline for control, administered once per day for 5 days. The cure rate was established by the assessment of four serial samples collected following treatment The cure rate was 3 of 18 quarters (16.7%) in the phage-treated group, whereas none of the 20 saline-treated quarters were cured which were already infected with *S. aureus*. Phage-infused healthy quarters continued to shed viable bacteriophage into the milk for up to 36 h

A lethal dose of *S. aureus* A170 was given to mice; phage MSa rescued 97% of mice and completely eradicated bacteria in vivo within 4 days

The phage MSa, delivered inside macrophages by *S. aureus*, kills the

Phage MSa was well tolerated by the animals, it drastically reduced inflammation, and it did not stimulate the production of neutralizing

Three of the bacteriophage isolates, phage/CIRG/1, phage/CIRG/4 and phage/CIRG/5, exhibited lytic activity against over 80% of the staphylococcal isolates. All isolates were stable up to 3 months at 37°C, and for 16 months at 4°C but the stability of their respective endolysins only lasted for 12–23 days at 37°C and 6 months at 4°C.

Authors analyzed *in vitro* the sensibility to phage infection of five *S. aureus* strains with drug resistance. Phages were stable at wide

In a mouse model of mastitis, chimeric SA2-E-Lyso-SH3B and SA2-E-LysK-SH3B reduce *S. aureus* CFUs by 1–3 log units in cow milk and by 0.63–0.8 log units in mammary glands. Synergism with lysostaphin

SA phage efficiently reduced bacterial growth in the bacterial

of phage treatment; when applied to nonlethal (5 × 106

intracellular staphylococci *in vivo* and *in vitro*

Lytic activity was determined *in vitro*

temperature and pH ranges

reduced CFUs by 3.36 log units

reduction assay

10-day infection, the phage also fully cleared the bacteria

Isolated phage was capable of infecting a wide spectrum of staphylococcal strains of both human and bovine origin

Bacteriophages

acute toxic effects

Study of bacteriophage

Isolation of a novel virulent bacteriophage (MSA6) from a cow

Isolation of bacteriophages virulent against *Staphylococcus aureus* associated with goat mastitis. Bacteriophages were

Isolation of a phage that infects *S. aureus* from bovine mastitis. SA phage was isolated from sewage

Fusion of endopeptidase domain from streptococcal endolysin SA2 with either lysostaphin or LysK endolysin and the recognition domain of endolysin LysK

with mastitis

isolated from soil and fecal samples

water

Endolysins

(MSa) active against *Staphylococcus aureus*, including methicillinresistant staphylococcal strains

Use of phage K to treat cow with subclinical mastitis. Twenty-four lactating Holstein cows with preexisting subclinical *S. aureus* mastitis were treated. Prior to experimentation with dairy cows, the phage preparations were screened in mice to determine

190 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

**Reference**

[54]

[68]

[69]

[70]

[71]

[72]

CFU/mouse)

#### **8. Animal models for treatment of other** *S. aureus* **infections**

Animal models have been widely used to evaluate the performance of phage therapy in the treatment of a variety of infections caused by *S. aureus*, usually nosocomial infections in humans. **Table 4** presents the use of phages and/or their endolysins in infections by *S. aureus* in animal models.



**Table 4.** Use of phages and endolysins against *S. aureus* infections using animals models.
