**4. Molecular epidemiology of** *S. aureus* **CC398**

The emergence of LA-MRSA strains in humans [16, 17, 25, 56] and the presence of an identical MRSA CC398 in pigs, farm workers, veterinarians who attended to the same pig farms and their nonexposed family members [47, 48] suggests animal-to-human or human-to-animal transmission. In an interesting report from the Netherlands, it was shown that farm visitors were positive for CC398 MRSA directly after a farm visit but tested MRSA negative after 24 h [19, 41]. These and other studies indicate that CC398 appears to be frequently shared between animals and humans and is capable of causing infections in both species [25, 70, 164]. Transmission of MRSA between animals and humans is not new, but the MRSA isolates, in most cases, represent an initial human-to-animal transmission [24, 49, 165, 166].

Analysis of MRSA and MSSA from animals and humans spanning 19 countries and four continents indicated that the CC398 lineage originated in humans as an MSSA [167]. The wholegenome sequencing analysis by Price et al. [167] demonstrated that livestock-associated MRSA CC398 lost an immune-evasion cluster (IEC) as it evolved from its human-adapted MSSA. All of the HA-MSSA strains carry ØSa3 prophage in association with human innate immunomodulatory genes that play crucial roles in human niche adaptation (**Figure 2**) [167]. The prophageassociated virulence and adaptation genes are not necessary for nonhuman hosts, therefore, ØSa3 is mostly absent in livestock strains. After their introduction to livestock, MSSA CC398 acquired resistance to methicillin and tetracycline. Since tetracycline is heavily used in animal farming, the tetracycline resistance gene *tet*(M) is nearly universal among livestock-associated MRSA CC398 and MSSA isolates. The MSSA and MRSA CC398 isolates found in humans with direct livestock contact exhibited the same molecular patterns (i.e., ØSa3 prophage negative, *tet*(M)-positive) as the livestock-associated strains, indicating human re-adaptation [167], and were also reported in isolates epidemiologically associated with human-to-human transmission in multiple countries and continents [168]. During the host jump from humans to animals, MRSA CC398 strains also acquired resistance to copper and zinc because of their use in animal feed [130]. The vast majority of LA-MRSA CC398 strains carry SCC*mec* type Vc, which contains the *czrC* gene that confers resistance to copper and zinc.

**Figure 2.** Gain or loss of genes as *S. aureus* jumps from human to livestock animals.

It is quite possible that LA-MRSA CC398 strains would eventually acquire certain genetic traits (additional antibiotic resistance and virulence factors) that would allow *S. aureus* to colonize both hosts and become a more formidable zoonotic agent [167]. Since ST398 strains are deficient in one or more restriction modification systems [169, 170], this adaptation process may have already occurred, as *pvl*-positive ST398 MRSA strains have been isolated from severe cases of community-acquired infections [20, 21]. These *pvl*-positive ST398 MRSA strains are also lysogenized with φSa3 phage coding for the human-specific virulence factors *sak, scn*, and *chp*. LA-MRSA *S. aureus* CC398 is not alone in the ability to adapt. A phylogenetic study of *S. aureus* CC97 strains that originated from livestock and caused human infections in Asia, Europe, and North and South America indicated that in the process of adapting to the human host, CC97 acquired either the SCC*mec* IV or V cassette and the φSa3 containing an immune evasion cluster [123]. A more recent study in the United States demonstrated that 22% of 30 veterinary students who were initially MRSA negative became positive after visiting MRSA-positive pig farms in Iowa but were negative again by 24 h after the visit [75]. The predominant *spa* type most commonly detected among the students was associated with ST5, suggesting a possible expansion of LA-MRSA to include ST5 [75].

Bayesian phylogenetic analysis of the poultry isolates with the CC5 clade indicated that it arose due to a single human-to-poultry host jump in or near Poland, where CC5 poultry strains acquired an MGE (mobile genetic element), presumably from other resident staphylococcal strains [36]. In addition to gene acquisition, loss of staphylococcal protein A (SpA) has occurred in avian isolates; it encodes virulence factors involved in human disease pathogenesis but is not needed for avian pathogenesis. The lack of protein A expression is a characteristic of the poultry biotype as defined by Devriese et al. [148]. In addition to the CC5 poultry clade, several other poultry isolates had identical or closely related STs to strains commonly associated with humans but had acquired MGE unique to avian strains, indicating that human-to-poultry host switches may be happening relatively frequently. A recent study demonstrated frequent contamination of poultry meat products with *S. aureus* ST5 and ST398 isolates, both of which have human origin [171]. Host adaptation of ST5 from humans to chickens was associated with a loss of genes contributing to human pathogenesis, and this was followed by the acquisition of avian-specific virulence determinants [36, 172]. Further, the presence of a cysteine protease encoded by a plasmid and widely distributed within avian strains suggests its potential role in avian-specific pathogenesis [173]. The adaptation from one host to another appears to be dependent upon the acquisition or loss of MGEs that code for key elements necessary for survival in new host [167, 174]. These cases have not only been associated with livestock [19] but have also occurred in cases with no known livestock contact [53, 54], suggesting a broader transmissibility capacity than originally thought. Further research is required to characterize the full scope of the genetic changes associated with the shift from humans to livestock and vice versa.
