**1. Introduction**

#### **1.1.** *Staphylococcus aureus* **as a zoonotic pathogen**

Besides infecting human hosts in hospital-acquired (HA), in community-acquired (CA) infections as an opportunistic pathogen and in food poisoning by enterotoxic strains, *S. aureus* has also been isolated from animal hosts, both in livestock-associated (LA) and in companion animals' infections. Due to the raise of methicillin-resistant *S. aureus* (MRSA) strains, this feature was included as a phenotypic marker to identify *S. aureus*, and now they are described as MRSA or methicillin-sensitive *S. aureus* (MSSA). Molecular epidemiology approaches helped to the


Modified from Refs. [5, 59, 60]; a [61]; b [62]; c [63]; d[64]; e ST deduced from homology between pet and human strains by PFGE and by *spa*-typing [65]; f [7]; g[8]; h[66]; i [67].

**Table 1.** Animal-associated genetic lineages of *S. aureus*.

understanding of the genetic structure of the *S. aureus* genetic population dynamics and hence in making predictions on transmissions between humans and animals. Multilocus sequence typing (MLST) is one of those molecular approaches. MLST analyzes the allelic combination of seven-to-nine (in *S. aureus* and other bacterial species) housekeeping genes that are randomly distributed along the genome. Mutations in *S. aureus* genes (*arcC, aroE, glpF, gmk, pta, tpi* and *yqiL*) are registered in an open public database (http://saureus.mlst.net) hosted at the Imperial College of London and supported by the Wellcome Trust Foundation. Each allele for each gene is designated with a specific number, so the allelic profile of a strain is designated by the numbers of alleles designated for each gene in the order described previously. Each allelic profile is designated with a sequence type (ST) number. STs sharing six or less alleles are grouped in clonal complexes (CC) in which the STs with the highest frequencies and number of shared alleles are designated as founder or subfounder clones, giving the name to the CC or related subgroups [1, 2]. Genetic lineages represented by a particular ST or CC are associated with specific hosts and geographical distributions. Some of them were originally described as specific for human or animal hosts and further reports associated them with animal or human transmissions, respectively, thus suggesting the zoonotic potential of *S. aureus* lineages. **Table 1** shows the major genetic lineages of *S. aureus* associated with animal hosts.

**1. Introduction**

180 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

**Genetic line‐**

**Original descri‐ bed host**

ST1 Human Cow, horse, chicken,

ST9 Pig Chicken –

ST121 Human Rabbit – CC126 Cow – –

CC705 Cow – – CC385 Chicken Wild birds – ST398 Pig Human, cow, chicken,

ST425 Cow – – ST1464 Sheep – –

**Table 1.** Animal-associated genetic lineages of *S. aureus*.

Modified from Refs. [5, 59, 60]; a

PFGE and by *spa*-typing [65]; f

horse, dogc

[61]; b [62]; c

[7]; g[8]; h[66]; i

CC133 Sheep Goat, cow, catb

piga

**age**

**1.1.** *Staphylococcus aureus* **as a zoonotic pathogen**

Besides infecting human hosts in hospital-acquired (HA), in community-acquired (CA) infections as an opportunistic pathogen and in food poisoning by enterotoxic strains, *S. aureus* has also been isolated from animal hosts, both in livestock-associated (LA) and in companion animals' infections. Due to the raise of methicillin-resistant *S. aureus* (MRSA) strains, this feature was included as a phenotypic marker to identify *S. aureus*, and now they are described as MRSA or methicillin-sensitive *S. aureus* (MSSA). Molecular epidemiology approaches helped to the

**Further reports Other features**

–

CC97 Cow Human, pigf,g Loss and acquisition of virulence gene and pathogenicity is-

ST239 Human Cow HA clone in Europe; isolates from bovine milk in Turkeyi

lands lead to change in host specificity; recent transmission be-

Acquisition of genetic elements to evade immune response in new hosts. *mecA*LGA251 (*mecC*); Spanish kennel dogs isolates

ST deduced from homology between pet and human strains by

tween cattle and pigs in Slovenia and Italy

, dogc Cat isolates from Japan; dog isolates from Spain

CC5 Human Chicken, turkey, dogb,c ST5. Major HA clone; dog isolates in Japan and Spain

ST8 Human Horse, cow, fishd USA300. Major CA clone; fish isolates in Japan

ST22 Human Cat, doge,c EMRSA-15 global CA epidemic clone

CC130 Cow Sheep, deerh In semiextensive red deer farm in Spain

[63]; d[64]; e

[67].

It is important to establish that the original description of a genetic lineage associated with a particular host followed by posterior reports of association with other hosts may not represent the evolutionary story of that lineage; it may only represent the original interest for the host due to the anthropocentric reasons or by the importance of the animal host as a food source or its contact with the human owner.

ST398 is one of the most reviewed cases of a clone showing animal-to-human transmission. Due to the whole-genome sequencing of strains from human endocarditis and bovine mastitis, differences in genomic content suggested that ST398 may be originated in humans. By loss, acquisition and reacquisition of pathogenicity islands or a staphylococcal chromosomal cassette related to methicillin resistance (SCC*mec*), and particular virulence genes like those encoding Panton-Valentine leukocidin (PVL) or the tetracycline resistance gene *tetR*, ST398 susceptible to methicillin was originally transmitted from humans to animals and then back to humans as a methicillin-resistant strain [3, 4]. Similar events may occur for bovine-specific clones from CC97. Staphylococcal protein A (*spa*) and clumping factor A (*clfA*), which are important in human pathogenesis, appear as nonfunctional mutants in bovine isolates, suggesting that they are not important for bovine colonization. Alleles of von Willebrand factor are specific for each host, and pathogenicity islands seem also specific for each host [5,6].

Reports of interspecies transmission of *S. aureus* infections are becoming more frequent. In a study of CA-MRSA distribution in Slovenia, ST398, an originally pig-associated genotype, was found in 9.9% of the cases [7]. CC97 was first described as associated with bovine mastitis cases and now has also been found in humans and pigs. Of particular interest is the case of a multidrug-resistant LA-MRSA genotype from Italy that has been transmitted to pigs as MSSA and spilled back after methicillin resistance acquisition [8]. Ovine-associated *S. aureus* isolates are represented by CC133. In a global survey in Western Europe and Mediterranean countries, CC700 and CC522 were also ovine-associated. This distribution differs from North and South America and Australia, where CC133 is the major ovine clone. Isolates from CC97 (bovineassociated), CC5, CC8 and CC30 (human-associated) were also found in this report, indicating high interspecific transmission of these genotypes [9]. Among zoological park animals in Greece, human-associated lineages ST80, ST8 and ST15, some of them with human pulsotype by PFGE analysis, suggest human-to-animal transmission [10]. ST80 and ST15 genetic lineages were also found in companion animals with close human contact in a veterinary teaching hospital in Greece. Panton-Valentine leukocidin (PVL), a necrotic toxin involved in skin infection, was found in 68.2% of MSSA isolates and in 50% of MRSA isolates, reinforcing the probable human origin of those strains. Also ST398 MRSA isolates were found that belong to the human cluster [11]. *S. aureus* has also been associated with wildlife animals. Studies in Spain demonstrate the presence of ST398 (pig- and human-associated) and ST1 (humanassociated) MRSA isolates harboring the novel *mecC* methicillin resistance gene (see below) in either red deer, Iberian ibex, wild boar or Eurasian griffon vulture, suggesting a probable human origin of these isolates [12–14]. All of these examples represent the high transmission capability of apparently species-specific *S. aureus* genetic lineages and urge to the implementation of both molecular epidemiology surveillance and novel infection controls.

Antibiotic resistance is also a major problem of *S. aureus* infections. There is a constant interchange of mobile genetic elements modifying the virulence arsenal of *S. aureus* genetic lineages. This suggests that genetic background may be considered for the design of modern strategies to control *S. aureus* infections.

After the discovery of penicillin by Alexander Fleming in 1928 and its application to treat *S. aureus* infections in 1940, the first penicillin-resistant *S. aureus* strains were reported by 1945. Later in 1959, methicillin appears as an alternative to the use of penicillin. By 1961, the first methicillin-resistant *S. aureus* (MRSA) strains were reported. A similar story occurred for vancomycin-resistant (VRSA) and vancomycin-intermediate (VISA) *S. aureus*. Methicillin resistance is encoded by a staphylococcal chromosome cassette named SCC*mec* containing the *mecA* or *mecC* (*mecA*LGA251) genes conferring resistance in humans and animals, for which at least 11 variants have been described. Apparently, these cassettes originated from a macrococcal *mecB* gene, which originated *mecA* (SCC*mec* and chromosomal forms) and *mecC* in staphylococci [15]. *mecC* has been almost exclusively associated with SCC*mec* type XI and located in animal strains from different STs and CCs [16], suggesting an intense intergeneric mobilization of SCC*mec* cassettes. VRSA strains seem not to be a major problem since only a dozen of clinical strains has been reported in the last decade. Vancomycin resistance is mediated by a complex of four genes (*vanA, vanH, vanX, vanY*) carried in a transposon. These modify a D-alanyl residue to D-lactate rendering the peptidoglycan structure resistant to vancomycin binding. *vanA* plasmids have also been reported, one of them being efficiently transmissible. This may predict that in the future, VRSA will also become a public health problem. Spontaneous mutants giving raise to VISA clones within vancomycin-susceptible *S. aureus* (VSSA) populations are known as heterogeneous-VISA (hVISA). hVISA/VISA is difficult to detect because on a first screening isolates behave as VSSA. Under the presence of vancomycin, VISA individuals are selected, and on a second screening, they behave as VISA. hVISA/VISA phenotypes have been associated with mutations in around 20 different genes that divert metabolism to peptidoglycan synthesis. Peptidoglycan then entraps vancomycin. hVISA/VISA reports are becoming more frequent in the literature, and it is to date considered of more relevance than VRSA. Staphylococci also present multidrug resistance genes such as *erm* (conferring resistance to macrolides, lincosamides and streptogramin B—MLSB-) and *vga* (conferring resistance to lincosamides, pleuromutilins and streptogramin A) genes. Some of these genes are located in plasmids or transposons that are highly mobile genetic elements [16]. All of these evidences suggest that antibiotic resistance is becoming a major public health problem for the control of *S. aureus* infections, so alternative biotechnological approaches different from classical antibiotic treatments must be used in the future to control *S. aureus* infections. Bacteriophage therapy is one of those approaches.

#### **1.2. Bacteriophages**

America and Australia, where CC133 is the major ovine clone. Isolates from CC97 (bovineassociated), CC5, CC8 and CC30 (human-associated) were also found in this report, indicating high interspecific transmission of these genotypes [9]. Among zoological park animals in Greece, human-associated lineages ST80, ST8 and ST15, some of them with human pulsotype by PFGE analysis, suggest human-to-animal transmission [10]. ST80 and ST15 genetic lineages were also found in companion animals with close human contact in a veterinary teaching hospital in Greece. Panton-Valentine leukocidin (PVL), a necrotic toxin involved in skin infection, was found in 68.2% of MSSA isolates and in 50% of MRSA isolates, reinforcing the probable human origin of those strains. Also ST398 MRSA isolates were found that belong to the human cluster [11]. *S. aureus* has also been associated with wildlife animals. Studies in Spain demonstrate the presence of ST398 (pig- and human-associated) and ST1 (humanassociated) MRSA isolates harboring the novel *mecC* methicillin resistance gene (see below) in either red deer, Iberian ibex, wild boar or Eurasian griffon vulture, suggesting a probable human origin of these isolates [12–14]. All of these examples represent the high transmission capability of apparently species-specific *S. aureus* genetic lineages and urge to the implemen-

tation of both molecular epidemiology surveillance and novel infection controls.

strategies to control *S. aureus* infections.

182 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

Antibiotic resistance is also a major problem of *S. aureus* infections. There is a constant interchange of mobile genetic elements modifying the virulence arsenal of *S. aureus* genetic lineages. This suggests that genetic background may be considered for the design of modern

After the discovery of penicillin by Alexander Fleming in 1928 and its application to treat *S. aureus* infections in 1940, the first penicillin-resistant *S. aureus* strains were reported by 1945. Later in 1959, methicillin appears as an alternative to the use of penicillin. By 1961, the first methicillin-resistant *S. aureus* (MRSA) strains were reported. A similar story occurred for vancomycin-resistant (VRSA) and vancomycin-intermediate (VISA) *S. aureus*. Methicillin resistance is encoded by a staphylococcal chromosome cassette named SCC*mec* containing the *mecA* or *mecC* (*mecA*LGA251) genes conferring resistance in humans and animals, for which at least 11 variants have been described. Apparently, these cassettes originated from a macrococcal *mecB* gene, which originated *mecA* (SCC*mec* and chromosomal forms) and *mecC* in staphylococci [15]. *mecC* has been almost exclusively associated with SCC*mec* type XI and located in animal strains from different STs and CCs [16], suggesting an intense intergeneric mobilization of SCC*mec* cassettes. VRSA strains seem not to be a major problem since only a dozen of clinical strains has been reported in the last decade. Vancomycin resistance is mediated by a complex of four genes (*vanA, vanH, vanX, vanY*) carried in a transposon. These modify a D-alanyl residue to D-lactate rendering the peptidoglycan structure resistant to vancomycin binding. *vanA* plasmids have also been reported, one of them being efficiently transmissible. This may predict that in the future, VRSA will also become a public health problem. Spontaneous mutants giving raise to VISA clones within vancomycin-susceptible *S. aureus* (VSSA) populations are known as heterogeneous-VISA (hVISA). hVISA/VISA is difficult to detect because on a first screening isolates behave as VSSA. Under the presence of vancomycin, VISA individuals are selected, and on a second screening, they behave as VISA. hVISA/VISA phenotypes have been associated with mutations in around 20 different genes

Bacteriophages are viruses that infect only bacteria. They coevolve with their hosts optimizing its spread and release mechanisms from the bacterial cell to the environment and cause (in the case of lytic bacteriophages) lysis of the bacteria. They are also a major driving force in *S. aureus* evolution as a pathogen since many virulence genes are mobilized between different strains by means of transduction [17]. Bacteriophages are the most abundant biological entities of nature, although they are present in all environments, it is in aquatic systems where they are in greater proportion [18, 19]. Early indications of the presence of viral particles were reported in 1896 when bacteriologist Ernest Hanking observed that from the waters of the river Jumma in India, they identify a "substance" with antimicrobial activity against *Vibrio cholerae* and this substance was also heat labile and capable of passing through the filters of porcelain used at that time [20]. Two years later in 1898 Gamaleya observed a similar phenomenon in *Bacillus subtilis*. In 1915 and 1917, Twort and D'Herelle, respectively, discovered the viral particles called bacteriophages [21]. Frederick Twort in 1915 reported antimicrobial activity against *Staphylococcus aureus* suggesting that it could be viral particles among other possibilities. As of D'Herelle, he coined the term bacteriophage in 1917; this discovery was due to their previous studies to develop a vaccine against dysentery where he observed lytic plaques later named as bacteriophages [22]. In 1923, the National Institute of Bacteriophages in Tbilisi Georgia was established. Since then, the search for lytic bacteriophages for the biological control of infectious diseases has been in the scene.

#### **1.3. Generalities**

Bacterial viruses (bacteriophages or phages) possess genetic material in the form of DNA or RNA; morphologically, they consist of a head and a tail both constituted of protein. The head is the core package of nucleic acid surrounded by a protein shell or capsid also called lipoprotein. The tail varies on complexity from one bacteriophage type to another [23]. According to their lytic activity, they can be divided into two groups: lytic and lysogenic bacteriophages. When bacteriophages infect their host, they reproduce and the process ends with lysis of the bacteria and release of viral progeny. This is known as the lytic cycle. When the bacteriophages are able to integrate its genetic material into the bacterial genome and thus reproduce for several generations together with their host's genome, they are called temperate phages and

they reproduce by a lysogenic cycle [24]. Bacteriophages which possess double-strand DNA express highly specific enzymes called viral-associated peptidoglycan hydrolyses (VAPGH) that bind to the bacterial cell surface and cause disruption of the cell wall to inject their DNA into the host cell [25]. The filamentous phage releases their viral progeny without causing the death of the bacteria [18], while nonfilamentous phages cause bacterial lysis by synthesizing endolysins (enzymes encoded by double-strand DNA phages) that hydrolyze peptidoglycan as part of an holin-endolysin system. The endolysins and holins are synthesized at late stages of phage infection. Endolysins accumulate in the cytoplasm until viral particles are assembled and holins form pores in the membrane allowing cytoplasmic translocation of endolysins through the membrane for peptidoglycan degradation [26]. Furthermore, single-stranded DNA or RNA bacteriophages synthesize "lysines" which interfere or inhibit the synthesis of the bacterial peptidoglycan [27]. The VAPGHs and endolysins are able to degrade the peptidoglycan when applied externally, which is why these enzymes represent an alternative to be used as enzybiotics in Gram-positive bacteria [28]. Bacteriophages and their endolysins are highly specific, infecting or hydrolyzing only a single species of bacteria attaching to specific receptors on the surface of host cell. The specificity of interaction between phage attachment structures and host cell surface receptors determinates host range. [29].
