Characterization of Virulence Factors in Enterotoxin-Producing Staphylococcus aureus from Bulk Tank Milk

Simple Summary Staphylococcus aureus, apathogen that causes bovine mastitis, produces various virulence factors, and human consumption of milk contaminated with the S. aureus enterotoxin may pose a public health risk. This study analyzed the genetic characteristics of bovine-mastitis-related virulence factors to evaluate the potential pathogenesis of S. aureus isolated from bulk tank milk. The results show that S. aureus isolated from bulk tank milk, not from mastitis, had a high prevalence of virulence factors and that the high presence of enterotoxins may be due to poor hygiene. Therefore, developing a strong monitoring and sanitation program for dairy factories is important to ensure hygienic milk production. Abstract Staphylococcus aureus, a persistent mastitis-causing pathogen, produces various virulence factors, including enterotoxins. This study analyzed the genetic characteristics of bovine-mastitis-related virulence factors to evaluate the potential pathogenesis of S. aureus isolated from bulk tank milk. Among 93 S. aureus isolates from 396 dairy farms operated by 3 dairy companies in Korea, 40 (43.0%) isolates carried one or more enterotoxin genes. Moreover, S. aureus carrying enterotoxin genes showed a higher prevalence in all virulence genes tested in this study except for pvl and lukM, which were not detected in any isolate, than in the isolates without enterotoxin genes. In particular, the prevalence of six genes (hla, hlb, lukED, fnbA, clfA, and clfB) was significantly higher in S. aureus carrying the enterotoxin genes than in the isolates without the enterotoxin genes (p < 0.05). The most common multilocus sequence type of enterotoxin-producing isolates was ST188, and all isolates of ST188 harbored the see gene. S. aureus isolated from bulk tank milk, not from mastitis, had a high prevalence of virulence factors, posing a public health threat. Moreover, a high presence of enterotoxins in bulk tank milk is probably because of poor hygiene; therefore, it is important to develop strong monitoring and sanitation programs for dairy factories.


Introduction
Staphylococcus aureus (S. aureus) is one of the most common pathogens causing contagious mastitis in the dairy industry [1]. In particular, S. aureus is persistent and causes chronic mastitis, and consumption of these dairy products transmits virulence factors from the contaminated milk to humans and may pose a public health risk [2,3].
S. aureus has various virulence factors, such as toxic shock syndrome toxin-1 (TSST-1), enterotoxins, and leukotoxins. In particular, the staphylococcal enterotoxin, belonging to the superantigen family, is the most potent because it can induce polyclonal activation of T cells at picomolar concentrations [4]. This activity is suspected to enhance virulence by inhibiting the host response to staphylococcal antigens produced during infection or present during toxinoses. Moreover, ingestion of enterotoxins causes severe food poisoning with vomiting, nausea, and diarrhea [5]. To date, more than 20 types of enterotoxins have been identified, of which classical enterotoxin types (sea-see) pose serious public health concerns because they

Molecular Typing
The genetic relationship of S. aureus with one or more enterotoxins was analyzed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). MLST was performed as previously described by Saunders and Holmes (2014) [20], and seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience, Freiburg, Germany) were sequenced with an automatic sequencer (Cosmogenetech, Deajeon, Korea). Sequence types (STs) were obtained by combination using the S. aureus database (https://pubmlst.org/ organisms/staphylococcus-aureus (accessed on 22 January 2022)). Moreover, PFGE was conducted by digesting genomic DNA using the SmaI enzyme (Takara Bio Inc., Shiga, Japan) according to a standard protocol of the Centers for Disease Control and Prevention (CDC, USA) [21], using a CHEF-MAPPER apparatus (Bio-Rad Laboratories, Hercules, CA), as described previously [22], and analyzed using the BioNumerics software (Applied Maths, Kortrijk, Belgium).

Statistical Analysis
The Statistical Package for the Social Sciences (SPSS) v.25 (IBM Corp., Armonk, NY, USA) was used for statistical analyses. Pearson's chi-squared and Fisher's exact test with Bonferroni correction were performed. Differences were considered significant at p < 0.05.
The distribution of virulence gene patterns in 40 S. aureus isolates carrying the enterotoxin genes is shown in Table 2. Although 19 virulence gene patterns showed no significant differences between the prevalence rate, S. aureus isolates carrying 8 virulence genes simultaneously were found in factory A1, which showed the highest prevalence of enterotoxin genes.

Distribution of Virulence Genes
The distribution of virulence genes in 93 S. aureus isolates is shown in Figure 2. The prevalence of virulence genes showed the difference depending on the presence of enterotoxin genes. In other words, S. aureus-carrying enterotoxin genes showed a higher prevalence in all virulence genes, except for pvl and lukM, which were not detected in any S. aureus isolates, than in the isolates without the enterotoxin genes. Among S. aureus isolates carrying the enterotoxin genes, hla (100.0%) and hlb (100.0%) were highly prevalent, followed by lukED (95.0%), fnbA (92.5%), clfA (50.0%), fnbB (47.5%), clfB (37.5%), icaD (35.0%), and icaA (15.0%). Moreover, the prevalence of six genes (hla, hlb, lukED, fnbA, clfA, and clfB) was significantly higher in S. aureus-carrying enterotoxin genes than in those without the enterotoxin genes (p < 0.05).   The distribution of virulence gene patterns in 40 S. aureus isolates carrying the enterotoxin genes is shown in Table 2. Although 19 virulence gene patterns showed no significant differences between the prevalence rate, S. aureus isolates carrying 8 virulence genes simultaneously were found in factory A1, which showed the highest prevalence of enterotoxin genes.

Genotypic Characteristics of S. aureus Carrying the Enterotoxin Genes
The genetic relationship of 40 S. aureus isolates carrying the enterotoxin genes is shown in Figure 3. Although PFGE revealed 29 clusters showing 85% similarity and no correlation with virulence factors, 5 STs were associated with virulence genes. In particular, none of the isolates of ST1 and ST72 carried the see gene; however, all isolates of ST188 harbored the see gene. Additionally, all isolates of ST1 carried the seh gene, whereas isolates of ST6, ST20, and ST72 did not harbor the seh gene. One isolate with both sea and sec genes and two isolates with the sed gene were only revealed in ST1. Although the relationship between ST and virulence factors, except for the enterotoxin genes, was not characterized in this study, all STs harbored hla and hlb genes; however, ST72 did not harbor fnbB, clfA, icaA, and icaD genes. relationship between ST and virulence factors, except for the enterotoxin genes, was not characterized in this study, all STs harbored hla and hlb genes; however, ST72 did not harbor fnbB, clfA, icaA, and icaD genes.

Discussion
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, most S. aureus toxins 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 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. Although 93 S. aureus isolates were from normal

Discussion
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, most S. aureus toxins 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 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. Although 93 S. aureus isolates were from normal bulk tank milk, not from mastitis, 40 (43.0%) carried at least 1 or more enterotoxin genes. Moreover, the presence of enterotoxin genes was significantly different among the factories. Interestingly, even in the same company, the prevalence of enterotoxin genes showed a significant difference among the factories. Previous studies reported the presence Animals 2022, 12, 301 7 of 10 of enterotoxins in 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 to be present at a higher rate in the enterotoxin-producing isolates than in 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][29][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, although none of the 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 that in the 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 the US (95.0%) [16,[33][34][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 of the isolates, the high prevalence of lukED in enterotoxin-producing isolates could imply 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 the 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 was significantly different. Previous studies have also reported a high prevalence of fnbA in S. aureus isolated from mastitis [29,39].
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 enterotoxin-producing and non-producing isolates carried tsst-1 in this study, S. aureus carrying the tsst-1 gene can lead to a public health concern.
In this study, five STs, ST1, ST6, ST20, ST72, and ST188, were revealed in enterotoxinproducing 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, possesses 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) 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. However, the bulk tank milk might not have been refrigerated to the correct temperature during transportation from the farms to the factories, or the temperature of whole milk might 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 mastitisrelated 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].

Conclusions
These data provide that S. aureus isolated from normal bulk tank milk, not from mastitis, has a high prevalence of enterotoxins and that S. aureus, which produces enterotoxins, has various virulence factors simultaneously, posing a public health threat. Moreover, the high presence of enterotoxins in bulk tank milk usually may be due to a combination of poor hygiene, bad milking technique, refrigeration failure, and unsanitary milking equipment. Therefore, developing a strong monitoring and sanitation program for dairy factories is important for hygienic milk production.