**2.3 Non-secretory factors**

Some of surface localized structural components that serve as virulence factors include membrane-bound proteins, which include collagen-binding protein, fibrinogen-binding protein, elastin-binding protein, penicillin-binding protein, and lipoteichoic acid. Similarly, cell wall-bound factors such as peptidoglycan, lipoteichoic acid, teichoic acid, protein A, β-Lactamase, and proteases serve as non-secretory virulence factors. Other cell surface-associated virulence factors include exopolysaccharides, which comprises capsule, slime, and biofilm. Overall, *S. aureus* has over 24 surface proteins and 13 secreted proteins that are involved in immune evasion [66] and about 15–26 proteins for biofilm formation [67, 68].

Surface proteins, such as staphylococcal protein A (SpA), clumping factors A and B (ClfA and ClfB) [69–71], fibrinogen-binding proteins [72], iron-regulated surface determinants (IsdA, IsdB, and IsdH) [69, 73], fibronectin-binding proteins A and B [74], biofilm associated protein (BAP) and exopolysaccharides (capsule, slime, and biofilms) [75–79], play roles in *S. aureus* adhesion to and invasion into host cells [80]. The BAP expression enhances biofilm production and the BAP gene is only found in *S. aureus* strain from bovine origin [81–83]. Evaluation of BAP gene of *S. aureus* from bovine and human isolates using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) showed that bovine and human isolates are not closely related [84]. Thus, some host-specific evolutionary factors may have been developed between both strain types.

Biofilms are considered an important virulence factor in the pathogenesis of bovine *S. aureus* mastitis [77, 78]. Slime, an extracellular polysaccharide layer, acts as a barrier against phagocytosis and antimicrobials. It also helps with adhesion to a surface [85]. If a biofilm forms in a mammary gland, it will protect those bacteria from antimicrobials and the host's immune system [77, 78]. In addition, once the biofilm matures and the immune attack has subsided, the biofilm can break open and allow reinfection of the mammary gland [86]. There are many contributors to biofilm production, such as polysaccharide intercellular adhesin (PIA) also known as poly-N-acetyl-β (1-6)-glucosamine (PNAG), MSCRAMMS, teichoic acids, and extracellular DNA (eDNA) [75, 76] that are known to help these bacteria cells to hold onto a surface [87]. Various proteins encoded by intercellular adhesion loci such as icaA, icaB, icaC, and icaD are involved in PIA production which in turn result in biofilm formation [75, 76]. Vasudevan et al. [88] evaluated the correlation of slime production and presence of the intercellular adhesion (*ica*) genes with biofilm production. These authors [88] found that all tested isolates were positive for *icaA* and *icaD* genes, and most tested isolates produce slime, but not all slime positives produced biofilms *in vitro*. Similarly, a study in Poland found that all isolates were positive for *icaA* and *icaD* [80] genes. While adhesion is promoted with biofilm production, the *bap* gene prevents the invasion of host cells [83]. Despite the presence of the *ica* gene strongly support biofilm production, the presence of

the *ica* gene is not mandatory for biofilm production since *S. aureus* lacking *ica* gene can still produce biofilm through other microbial surface components recognizing adhesive matrix molecules (MSCRAM) and secreted proteins [89, 90].

#### **2.4 Secretory factors**

Some of the known secretory virulence factors are toxins which include staphylococcal enterotoxins, non-enteric exfoliative toxins, toxic shock syndrome toxin 1, leucocidin, and hemolysins (alpha, beta, delta, and gamma) [91, 92]. Similarly, enzymes such as coagulase, staphylokinase, DNAase, phosphatase, lipase, phospholipase, and hyaluronidase serve as virulence factors of *S. aureus* [93].
