S. aureus produces many potential virulence factors to promote host tissue colonization, adhere to host cells, resist physical removal, invade host cells, and compete for iron and other nutrients [23]. In particular, plasmids and transposons typically contain antibiotic resistance genes, whereas phage-related and pathogenicity islands contain most S. aureus toxins and other virulence determinants [24]. Moreover, the most S. aureus toxin and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs) [24], and the transfer of virulence genes via SaPIs may increase the risk of pathogenicity because they are transmitted not only to the same species but also to the completely unrelated bacteria such as L. monocytogenes.
In Korea, five major dairy companies produce 84% of the total milk and dairy products (ATFIS, 2020) [25], and S. aureus isolated from six factories operated by three dairy companies was investigated in this study. Enterotoxins are a major cause of staphylococcal food poisoning, and classical enterotoxins account for 90% of foodborne illnesses [7]. Although 93 S. aureus isolates were from normal bulk tank milk, not from mastitis, 40 (43.0%) carried at least one or more enterotoxin genes. Moreover, the presence of enterotoxin genes was significantly different between factories. Interestingly, even in the same company, the prevalence of enterotoxin genes showed a significant difference among factories. Previous studies reported that enterotoxins found from 27.1–79.0% of S. aureus in milk and dairy products, and the frequency of enterotoxin genes varied by geographic region [13, 16, 26]. Furthermore, Schelin et al. (2011) [27] reported that enterotoxin production was influenced by environmental factors, such as temperature, pH, and moisture, therefore, management programs of dairy factories may affect the level of enterotoxins.
In the distribution of several virulence factors, which implicate the pathogenesis of S. aureus, all virulence genes, except pvl and lukM tested in this study, were shown at a higher rate in the enterotoxin-producing isolates than non-producing isolates. Primarily, hla and hlb genes allow for more persistence of pathogens in the mammary gland and cause chronic infection [6]. Therefore, these genes are the most prevalent in S. aureus from bovine mastitis milk [17, 28–30]. Moreover, enterotoxin-producing S. aureus from normal bulk tank milk in this study is highly likely to cause chronic mastitis.
Leukocidin, including PVL, a type of cytotoxin, is an important factor contributing to increased virulence [31, 32]. In this study, in contrast of none of S. aureus isolates carried pvl and lukM genes, 95.0% of enterotoxin-producing isolates carried the lukED gene, and the prevalence was significantly higher than non-producing isolates. Previous studies have reported that lukED was the most prevalent in S. aureus isolated from bovine mastitis milk in South Africa (100.0%), Finland (96.6%), Japan (96.0%), and US (95.0%) [16, 33–35]. The lukED has the ability to penetrate and kill cells, such as neutrophils that carry the bovine chemokine receptor, and is essential for the pathogenesis of mastitis [36]. Although pvl and lukM, which are associated with leukocyte destruction and necrosis and severity of mastitis, respectively, were not detected in any isolate, high prevalence of lukED in enterotoxin-producing isolates could infer that there is a potential to induce mastitis.
Adhesion is essential to invade host cells and evade immune responses [37], and the biofilm-forming ability causes chronic or persistent infections [38]. S. aureus harbors various adhesion and biofilm-related genes, such as fnbA, fnbB, clfA, clfB, icaA, and icaD, and prevalence of these genes has been reported to vary according to geographic regions [18]. In this study, the prevalence of fnbA (92.5% vs. 52.8%), clfA (50.0% vs. 20.8%), and clfB (37.5% vs. 3.8%) genes between enterotoxin-producing and non-producing isolates were significantly different. Previous studies have also reported a high prevalence of fnbA in S. aureus isolated from mastitis [28, 39].
Another superantigen virulence factor, tsst-1, hyperactivates the host immune response, resulting in toxic shock syndrome in humans, and can retain biological activity in milk after pasteurization [6]. Although each one among enterotoxin-producing and non-producing isolates carried tsst-1 in this study, S. aureus carrying tsst-1 gene can lead to a public health concern.
In this study, five STs, ST1, ST6, ST20, ST72, and ST188, were revealed in enterotoxin-producing isolates. Song et al. (2015) [40] reported that ST1, ST6 and ST188 are frequently found to be associated with staphylococcal food poisoning in East Asia. Wang et al. (2018) [41] also reported that S. aureus ST188, a major lineage-causing infection in humans and livestock, possess high nasal colonization and biofilm formation abilities in several host species. Mechesso et al. (2021) [42] have identified ST188 from bovine mastitis in Korea, but its prevalence was 23.3%. In this study, the prevalence of ST188 was higher than that of other STs, therefore, it seems to have a high potential to induce mastitis. Moreover, interestingly, all isolates in ST188 harbored the classical enterotoxin gene, see.
The see gene, which has the highest prevalence (75.0%) in enterotoxin-producing S. aureus in this study, has reported no or only low detection in milk [1, 13, 43]. Homsombat et al. (2021) [7] reported that the growth of see-positive staphylococci in milk was significantly faster at a temperature of more than 8°C. In this study, each bulk tank milk sample was collected from factories and sent to the laboratory under 4°C. But the bulk tank milk may not have been refrigerated to the correct temperature during transportation from farms to factories, or the temperature in whole milk may have risen during the milk in/out process.
Moreover, novel SEs, seg, seh, and sei genes were detected in 40.0%, 25.0%, and 20.0% of isolates, respectively, and have also been reported in food poisoning and bovine mastitis-related S. aureus worldwide [40, 42, 44, 45]. Although the mechanism of novel enterotoxins in S. aureus is not clearly known, several molecular studies have suggested that they may play an important role in enhancing virulence because they are widely distributed in S. aureus [46].