**2. Staphylococcal Enterotoxins (SEs)**

Some *S. aureus* strains are capable of producing SEs, and several CoNS species have also been reported to produce SEs, including *Staphylococcus epidermis* and *Staphylococcus haemolyticus*, among others [27–29]. Classified into members of the pyrogenic toxin superantigen family [30], SEs are a series of extracellular single-chain proteins primarily produced in food or culture media and secreted by some *Staphylococcus* strains, and the causative agent of SFP, which after ingestion may cause intoxication exhibited by vomiting (emetic action on the abdominal viscera) and diarrhea (epithelium of the intestinal tract and inhibition of water absorption in intestine), even collapse in severe cases [15]. In most SFP cases, a single enterotoxigenic staphylococcal strain isolated from the contaminated food is suspected to be the responsible strain [31]. However, from a number of SFP outbreaks with symptoms similar to gastrointes‐ tinal syndromes mediated by SEs, only nonenterotoxigenic staphylococci have been isolated, which may be explained by the outgrown enterotoxigenic staphylococci by the nonentero‐ toxigenic ones. Containing low α-helix and high β-pleated sheet content, SEs have similar and flexible structure, with a low molecular weight between 24,000 and 30,000 Da [32]. One of the characteristic features of SEs is their heat stability (withstanding heating to 121°C for 10 min), as SEs are commonly produced ranging from 10°C to 50°C, with the optimum at 30°C to 40°C [24, 26, 33–35]. Consequently, when *Staphylococcus* strains grow in food under appropriate conditions and produce SEs undetectable by taste or smell, SEs remain active even when bacteria itself have been eliminated by heating. As concluded, effective ways include the prevention of food contamination from staphylococci, staphylococci organisms from growing, and SEs from being produced under either heating or freezing. SEs are also highly hydrophilic, with pH ranges from 4.8 to 9.0 (optimum between 5.3 and 7.0) and water activity (aw) ranges from 0.87 to 0.99 (optimum at 0.90) [24, 36, 37]. Being well studied and documented, SEs are found to possess a number of biological properties, including superantigenicity (induction of T-cell mitogenicity and human interferon), emetic activity, and pyrogenicity [38]. In addition to gastrointestinal symptoms in SFP as aforementioned, SEs have also been implicated in other diseases such as atopic eczema [39–41], rheumatoid arthritis [41–43], and urticaria [41, 44]. With binding of functional SEs (not presursor) to both the α-helical regions of the major histocompatibility complex (MHC) class II molecules outside the peptide binding groove of the antigen-presenting cells (APC) and the variable region (Vβ) on the T-cell receptor (TCR), a bridge between T cells and APC is formed, leading to nonspecific activation and proliferation of a large number of T cells [41, 45, 46] resulting in robust inflammatory cytokine release.

As classified according to the distinct immunological entities, until recently, a total of 25 types of SEs (A–V and X, with 3 subtypes for C) have been identified. The finding of SEs has been in accordance with the development of identification methodologies. In early studies, animal testing experiments had been commonly used for the observation of SE activity, requiring monkey, feline, or cavy models [47–49]. After feeding with contaminated food, animals exhibited abnormal behavior or gross morphology changes, and the SEs contained in food were determined by the number of vomiting events, the time until the first vomiting event, and behavioral changes [47]. These types of animal feeding experiments had been commonly used for the characterization of emetic activities of SEs, thus determining the roles for emetic activities of SEs during SFP. However, such methodologies were also significantly limited by low sensitivity and specificity, poor reproducibility, high expense and laboratory operation, diversity in animals, and incapability of quantification and accurate identification [47]. Therefore, the identification of individual single type of SEs was reported by the availability of serological assays. Based on the specific reaction between antigen and antibody, identifica‐ tion of SEs via specific antibody [50] was first reported in the 1930s. Ouchterlony double immunodiffusion, also known as agar gel immunodiffusion, had become applicable in 1948 [51], and the first serological study on distinct SEs was conducted in 1958 [52]. From 1960s to 1970s, a number of SEs with emetic activities were identified and reported by the employment of serological methodologies including Ouchterlony double immunodiffusion, radial immu‐ nodiffusion, and enzyme-linked immunosorbent assays (ELISA). With the development of molecular biotechnology such as PCR and even genome sequencing in the 1990s and 2000s, a large number of newly identified SEs (G–V and X) as well as variants (for instance, 17 distinct variants of *sel-x*) had been discovered on enterotoxin gene cluster (*egc*), staphylococcal pathogenicity islands (SaPIs), mobile genetic elements (MGEs), and even the bacterial genome (*sel-p* on genome of N315).

#### **2.1. Classical SEs**

Although investigation on the observation and detection of SEs from animal feeding experi‐ ments could be dated back from 1930s, the immunological characteristics were not clarified until a number of serological studies had been conducted in the 1950s and 1960s. These studies revealed 6 types (A, B, C1, C2, D, and E) of SEs that were characterized and further referred to as classic SEs (including C3 reported in 1984). Antigenicity of SEs was validated when the antisera prepared from rabbits infected with SEs was demonstrated to protect cats [52], and later from a further serological study, the origins of two heat-resistant types of SEs had been verified [52, 53]. These extracellular immunologically distinct SEs responsible for the clinical manifestation of SFP were first referred to as type F (food poisoning) and type E (mostly produced by strains of "enteritis" origin) and later designated as A and B for a better sequential numbering system, with only type A commonly associated with food poisoning (A-1963). Sharing a similar basic three-dimensional structure, the 5 serological groups of SEs (A–E) exhibit nucleotide sequence identity of 50% to 85%, with types A, D, and E categorized into one group (52–83% amino acid identity) and types B and Cs falling into a separate, more closely related group (62–64% amino acid identity) [53, 54]. As origins and sources, types B and C are important causes of nonmenstrual-associated TSS, and types A and D are common causes of SFP with types B and C to a lesser extent [49].

#### *2.1.1. Staphylococcal Enterotoxin A (sea or SEA)*

SEA was first identified in 1959 from *S. aureus* strain FRI-196E [52, 55] and then named as type A [56], which has been considered to be the most commonly detected SE associated with food poisoning, with its minimal toxic dose for humans ranging from 20 to 100 ng [24]. Also, SEA has been verified to be responsible for a number of SFP outbreaks, including an extensive outbreak caused by ingestion of dairy products in Osaka, Japan, in 2000 [24, 56, 57]. After identification by serological methodology, the production of SEA in different media or condition had become a major concern. The maximal level of SEA production was found to occur during exponential phase. In semisolid BHI agar (pH 5.3], the production of SEA was acquired, and trace amounts of SEA and SEB were obtained with cellophane sac culture [58, 59]. As food samples were concerned, SEA production was detected in a number of meat samples (raw beef and pork, cooked beef and pork, and canned ham). Better growth of *S. aureus* and production of SEA were detected in cooked meat than raw meat despite no significant difference obtained, and such diversity may be explained by the bacterial competition between anaerobic and aerobic conditions (with the latter preferable for *S. aureus*) [50, 58, 59]. In milk, SEA production was also found to be associated with staphylococcal growth [60]. In fermented sausage, SEA production had been detected aerobically at pH 5.1 (with an inoculum of 4×107 cells/g sausage) but not anaerobically at pH 5.7 [61]. For co‐ growth food microorganisms, inhibition was observed to be more common than stimula‐ tion, including inhibition of both staphylococcal growth and SE formation with no apparent effect on growth [62]. However, such influence of other microorganisms on *Staphylococcus* was affected markedly by environmental conditions, and discrepancy on inhibition had been also noticed between plate test and meat slurries [58, 59]. Despite acquisition of appropri‐ ate staphylococcal growth in both pure culture and in the presence of other food microorgan‐ isms, production of SEA was only formed in pure culture [58, 59]. Although SEB was reported to be produced in much larger quantities and more diverse among strains than SEA, SEA had been implicated in a larger number of food poisoning cases [63]. Resembling a pri‐ mary metabolite (with SEB as a secondary metabolite), SEA is secreted by the bacterium during the exponential phase of growth, with various of factors affecting its production, including salt concentration (NaCl, NaNO2, and NaNO3 showing no influence), surfactants (increase in SEA secretion), pH (optimal ranging from 6.5 to 7.0), and antimicrobial agents (inhibition by chloramphenicol or 2,4-dinitrophenol, with streptomycin or penicillin G exhibiting no influence), which may explain the higher frequency and incidence of SEA in food poisoning [63–66]. In addition, temperature and inoculum size play important roles in SEA production. As temperature was concerned, SEA production was detected under a broad range of temperatures from 10°C to 50°C (in BHI broth) but not at low temperatures (such as 8°C or 10°C as reported) [24]. SEA was detected in the exponential phase from 15°C to 37°C, and its production increased with the elevation of temperature. Also, SEA was detected in the stationary or death phase at 10°C despite acquisition of the lowest SEA concentra‐ tion at this temperature. Similar to SEE, SEA contains 2 MHC-II binding sites (Zn2+ depend‐ ent) and thus possesses strong superantigenicity for T-cell activation [67, 68]. Carried by a polymorphic family of lysogenic phages [69, 70], the gene encoding SEA has a length of 771 bp, and its translational product is SEA precursor of 257 amino acids. With 24-residue Nterminal hydrophobic leader sequence further processed, the mature form of SEA was composed of 233 amino acids [38, 53, 68–74]. Unlike other classic SEs (*seb*, *sec*, and *sed*), expression of *sea* had been found to be independent of *agr* regulation [54, 75, 76].

#### *2.1.2. Staphylococcal Enterotoxin B (seb and SEB)*

Being the first identified (from *S. aureus* strain FRI-243) and the most studied SE, SEB was initially named as type E and subsequently designated as type B. As the most potent SE and requiring much lower quantities for toxic effect than synthetic chemicals, SEB are capable of causing multiorgan system failure and death at low concentration. As an exotoxin secreted by *S. aureus*, production of SEB had been reported from diverse clonal complexes, including CC8 (the most common), CC20, and CC59 [32, 77–79]. As a superantigen capable of cross-linking APCs and T cells to form a ternary complex between MHC-II and TCR at specific Vβ chain, SEB had been well studied as a causative agent for food poisoning, TSS, atopic dermatitis (common colonization of *S. aureus* and frequent occurrence of SEB-specific antibody from patients with AD), and respiratory diseases (asthma and nasal polyps) [32, 77, 80, 81]. As a well-characterized protein, SEB had been found to be extremely stable (retaining its activity even in acidic environment), water soluble, heat stable (among the most heat-stable proteins, with intact protein under 78°C to 80°C for 30 min), broadly pH tolerant [4–10], and resistant to proteolytic digestion (such as pepsin, trypsin, and papain) [47, 82, 83]. Nevertheless, SEB formation and production were influenced by a number of factors, including inhibition of SEB formation in BHI broth by medium filtrates (such as K2HPO4, KCl, CoCl2, NaF, acriflavine, phenethyl alcohol, streptomycin sulfate, chloramphenicol, spermine phosphate, spermidine phosphate, and Tween-80) [82], decrease of SEB production by either temperature depletion (without affecting staphylococcal growth) or curing salt concentration elevation (more rapid reduction of SEB production than staphylococcal growth) [84], catabolite repression [85], and minerals (double SEB production was obtained when magnesium and potassium are under appropriate concentration) [34, 86, 87]. Generally, maximal SEB production occurs in postex‐ ponential growth. From early studies in the 1950s and 1960s, SEB was considered to be irrelevant to food poisoning [50, 88]. Located in either chromosome (strain FRI-243, FRI-277, or S6) or plasmid (strain DU-4916), *seb* is 705 bp in length, and the mature SEB consists of 239 amino acid residues sharing nucleotide and amino acid sequence homology with *sec1* and streptococcal pyrogenic exotoxin A [89–91]. Regulated by the staphylococcal two-component system, accessory gene regulatory (*agr*), the region between 59 and 93 nucleotides upstream of the transcription ignition site was found to be essential for transcription and expression [92– 94]. *seb* had been commonly found in toxin-mediated foodborne and clinical *S. aureus* strains, and recently, *seb* (by PCR and Western blotting) was detected from 5% of 300 clinical *Staphy‐ lococcus* strains [95].

#### *2.1.3. Staphylococcal Enterotoxin C (sec and SEC)*

According to the new numbering system agreed on the American Society for Microbiology (ASM) meeting in 1962, the first verification of SEC was then reported in 1965, with its toxicity and specificity also confirmed [96]. In this study, the enterotoxins from *S. aureus* strains FRI-137 and FRI-361 were both discovered to react with a specific antibody; thus, strain FRI-137 (ATCC 19095) was selected as the prototype of SEC [96]. However, 2 years later, enterotoxins from strain FRI-137 and FRI-361 were purified as distinct enterotoxins [96, 98] and consequently labeled as SEC1 (strain FRI-137) and SEC2 (strain FRI-361). In 1984, the third enterotoxin C (SEC3) was discovered from a *S. aureus* strain FRI-913 from prawn in England, which were serologically and chemically similar to SEC1 and SEC2 but identical by isoelectric focusing, radioimmunoassay (RIA), and N-terminal analysis [99, 100]. Despite cross-reactivity with same antibody, each of the 3 SEC had antibodies that reacted with minor determinants [99]. Located on chromosome (SaPIs), *sec* is composed of 801 bp and encodes a precursor protein of 267 amino acids, with the mature toxin of 239 amino acids [101, 102]. Aside from 3 types of classic SECs, additional *sec* variants (such as *sec*-bovine from SaPIbov) possessing >95% deduced amino acid homology among them had been also reported [103–107]. As SEC1, SEC2, and SEC3 are all emetic enterotoxins with equal toxicity to that of SEA and SEB in both oral administration and intravenously [96, 99], SEC have been responsible for numerous SFP outbreaks (mostly caused by milk) [108]. Maximal production of SEC occurs during postex‐ ponential growth. SEC-positive strains of *S. aureus* are commonly associated with bovine, ovine, and caprine dairy products [109]. Yet interestingly, SEC expression has been noted to be reduced in cheeses [110]. From a recent study, milk environment was found to dramatically change the expression profiles of enterotoxin genes despite no influence on staphylococcal growth. In particular, SEC production was substantially reduced in milk compared to the laboratory medium on the protein level, which may be explained by the down-regulation of the *agr* system [111].

#### *2.1.4. Staphylococcal Enterotoxin D (sed and SED)*

In 1967, SED was first reported from *S. aureus* strain FRI-293 (which also produced SEC; thus, strain FRI-494 was selected as the prototype strain, also known as ATCC 23235) and its emetic activity in cats, and specific neutralization of biological activity by antisera had been verified [112]. The production of SED alone and in combination with SEA was considered to play a key role in food poisoning (ranking second in frequency after SEA) and recognized as one of the most commonly recovered enterotoxins in SFP outbreaks [112, 113]. Encoding a toxin of 228 amino acids, *sed* is located on a 27.6-kb penicillinase plasmid pIB485 [114]. SED was found to be partially activated by *agr* via RNA III-mediated reduction of Rot (repressor of toxin) during postexponential growth phase, as independent formation from *agr* was found under high concentration. As a consequence of *agr* regulation via quorum sensing, during growth in BHI broth, a modest postexponential induction ratio (<10-fold) was obtained as *sed* reached maximal production during transition from exponential to stationary phase of growth [113, 115]. With the existence of a consensus -10 sequence, a less conserved -35 sequence, and a TG dinucleotide motif, the presence of 52-bp sequence (from -34 to +18) and transcription from +1 to +18 were important for promoter function and *agr* regulation [116]. Aside from regulation by the *agr* system, NaCl stress was capable of decreasing *sed* expression, although no significant effect was further verified. However, regulation under NaCl stress may be highly strain specific variable [117]. As food samples were concerned, in cheese manufacturing (with starter culture including 103 CFU/ml of milk), *sed* expression was not induced even when inoculated at 106 CFU/ml (equal to 108 CFU/g of cheese), presenting a low level of expression and a prolonged pattern that was similar to SEA [113, 118]. In different ham products, when *S. aureus* was inoculated for optimal growth in cultivation broth for 7 days, continuous *sed* expression was observed throughout the entire incubation period for both boiled and smoked ham [115]. However, much less production of *sed* (9 times less) was detected in the latter. For Serrano ham, SED was only detected after 5 days of incubation (*sed* expression still too low to determine), similar to which a second increase had been obtained for boiled and smoked ham after the same time span of incubation [115].

#### *2.1.5. Staphylococcal Enterotoxin E (see and SEE)*

In 1971, SEE was reported from a food poisoning *S. aureus* strain FRI-326, which produced distinct SEE having no immunoreactivity with specific antibodies to other SEs [119]. Its toxicity (in rhesus monkeys), specificity, and neutralization with specific antibody were also validated [119]. Located on the phage, *see* is composed of 771 bp and encodes a precursor with a molecular weight of 29,358 Da, which was further processed to a mature extracellu‐ lar form with a molecular weight of 26,425 Da [120]. Containing a single polypeptide chain, SEE consists of 259 amino acid residues (no free sulfhydryl groups found), with serine and threonine as the NH2- and COH-terminal amino acids, respectively [121]. Under extreme acidic (pH 2) and basic (pH 12) conditions as well as heating, the toxicity (emetic activity) and antigenicity (serological activity) were found to be destroyed, which is likely due to conformational change [121].

#### **2.2. Staphylococcal enterotoxin-like toxins**

Before the 1990s, a total of 7 types of classic SEs (*sea*, *seb*, *sec1*, *sec2*, *sec3*, *sed*, and *see*) had been known as causative agents of SFP in humans due to emetic activity. However, starting from the discovery of *seh* in 1994 (aside from discovery of *sef* on 1981), a large variety of novel SE or SE-related toxins (as well as variants) had been reported (G–V and X) based on genetic homology with classical SEs. In 2004, the International Nomenclature Committee for Staphy‐ lococcal Superantigens has proposed that only staphylococcal superantigens inducing emesis after oral administration in a primate model should be designated as SEs, whereas other related toxins lacking either emetic properties in a primate model or verification of emetic activity should be otherwise designated as staphylococcal enterotoxin-like toxin type X [122, 123].

#### *2.2.1. Staphylococcal Enterotoxin-Like Toxin Type F (sel-f and SEl-F)*

In 1981, Bergdoll et al. had noticed an enterotoxin-like protein recovered from 93.8% [61/65] *S. aureus* strains sampled from patients with TSS, representing the first evidence of *sel-f* [124]. With its purification and preparation of specific antibody, *sel-f* was also recovered from 11.5% [3/26] of laboratory *S. aureus* strains, compared with only 4.6% [4/87] from other sources, which suggested the association between SEl-F and TSS [124]. However, from an investigation on the spread of a TSS strain, a temporal association of antibodies to SEl-F with cessation of recur‐ rences of TSS was found, indicating that its production may not either reach clinically significant levels during infection or is insufficient to cause TSS [125]. Generally, studies and reports on SEl-F have been rarely available.

#### *2.2.2. Staphylococcal Enterotoxin-Like Toxin Type G (sel-g and SEl-G)*

In 1998, SEl-G and SEl-I (from *S. aureus* strains FRI-572 and FRI-445, respectively) had been identified and characterized, including verification on emesis (eliciting emetic response in rhesus monkeys) and superantigenicity (proliferation of T cells) [126]. *sel-g* consists of 777 nucleotides and encodes a precursor protein of 258 amino acids, which has typical bacterial signal sequences and is then cleaved to form mature toxin with 233 amino acids and with a molecular weight of 27,043 Da [127, 128]. SEl-G showed higher homology to SpeA, SEB, SEC, and SSA (38–42% amino acid identity) and exhibited similar epitopes with SEC1 [126].

#### *2.2.3. Staphylococcal Enterotoxin-Like Toxin Type H (sel-h and SEl-H)*

In 1994, the first discovery of *sel-h* from *S. aureus* strain D4508 was reported, with its nucleotide and amino acid sequences identified [129]. One year later, SEl-H was identified and purified from *S. aureus* strain FRI-569, which elicited an emetic response in monkeys and was found to be antigenically distinct from other existent SEs [51]. SEl-H shares about 35% amino acid identity with SEA, SED, and SEE [130]. As a superantigen homologous to SEA subfamily, SEl-H displays unique MHC-II binding properties. As a potent T-cell mitogen, SEl-H was capable of activating large amounts of T cells by cross-linking APC and T cells via Vα domain (Vα10, TRAV27) of TCR (with no TCR Vβ-specific expansion) by direct interaction between SEl-H and TCR Vα domain [131, 132]. With emetic activity, *sel-h* was commonly detected alone or together with *sea* [133] and responsible for a number of SFP outbreaks. In 1996, an outbreak was caused by cheese and *S. aureus* strains isolated from cheese were found to produce SEl-H [133]. From the SFP outbreak caused by reconstituted milk in Japan, SEl-H was also detected along with SEA. In a survey on 146 *S. aureus* strains isolated from humans, cows, and bovine in Japan, 7 and 4 strains were found to harbor *sea*<sup>+</sup> *sel-h*<sup>+</sup> and *sel-h* alone, respectively [57, 133]. In December 2003, a suspected SFP outbreak involving 8 persons (3 adults and 5 children) with symptoms of vomiting, stomach cramps, and diarrhea shortly after lunch was caused by contaminated mashed potato, and *S. aureus* strains contained in raw bovine milk for prepara‐ tion of mashed potato were found to produce sufficient SEl-H for food poisoning [134]. SEl-H production was influenced by a variety of factors, including aeration and pH conditions. Higher production level of SEl-H was acquired under aerobic incubation or pH controlled at 7.0, with decrease in SEl-H production under anaerobic condition or slight change of pH (such as 6.5 or 7.5) [135].

#### *2.2.4. Staphylococcal Enterotoxin-Like Toxin Type I (sel-i and SEl-I)*

As aforementioned, SEl-I were identified together with SEl-G in 1998 [126]. Unlike SEl-G, SEl-I was more similar to SEA, SED, and SEE (26–28% amino acid identity). *sel-i* consists of 729 nucleotides and encodes a precursor protein of 242 amino acids, which contains typical bacterial signal sequences and is further cleaved to form mature SEl-I of predicted 218 amino acids with a molecular weight of 24,928 Da [127, 128]. Although separated by DNA related to other SEs, linkage of *sel-g* and *sel-i* was discovered, and this enterotoxin gene cluster was designated as *egc*, with *sel-g* located 2002 bp downstream of *sel-i* [127]. In southern France, carriage of *sel-g*<sup>+</sup> *sel-i*<sup>+</sup> and *sec*<sup>+</sup> *sel-g*<sup>+</sup> *sel-i*<sup>+</sup> was detected from 41.9% and 24.5% of 155 *S. aureus* strains isolated from various food samples [128]. In Taiwan, 14.5% [8/55] *S. aureus* strains of human origin and 9.4% [13/139] strains isolated from frozen food, Chinese sausage, and meal boxes were found to harbor *sel-g*, *sel-h*, and/or *sel-i*, suggesting a minor role that such SEs play in SFP outbreaks [136]. However, a discrepancy between the presence of *sel-g* and *sel-i* and the production of enough quantities of SEG and SEI was also noticed [128]. In 2004, 10.1% [11/109] wild *Staphylococcus* spp. stains were found to contain SEs and *egc*, and the *egc* from strain AB-8802 present variants of *sel-g* and *sel-i* (*sel-gv* and *sel-iv*) [137].

#### *2.2.5. Staphylococcal Enterotoxin-Like Toxin Type J (sel-j and SEl-J)*

In 1998, *sel-j* was first found to be located on the plasmid pIB485 encoding *sed*, which was separated from *sed* by 895 bp of intergenic region containing a perfect inverted repeat (with each arm of the repeat having a length of 21 bp) [138]. Most of *sel-j* was detected on *sed*encoding plasmid, suggesting the coexistence of these 2 SEs and their relative contribution to the food poisoning symptomology [138]. With transcription in opposite directions, both *sel-j* and *sed* were capable of expression in *S. aureus* strains, with *sed* only under the transcrip‐ tional control of *agr* [138]. Containing 269 amino acid residues, sequence of SEl-J showed substantial similarity to the SE family of *sea*, *sed*, and *see*.

## *2.2.6. Staphylococcal Enterotoxin-Like Toxin Type K (sel-k and SEl-K)*

Despite observation of the *sel-k* gene on SaPI1 (in 1998) and *egc* from *S. aureus* strains A900322 (in 2001), the first designation of *sel-k* from *S. aureus* TSS isolates MN NJ was reported in 2001, with its identification on SaPI3 together with *seb* [139, 140]. Possessing biochemical and biological properties similar to classic SEs, including superantigenicity (Vβ-specific T-cell activation), pyrogenicity, emesis, and lethality in primates, SEl-K was secreted by clinical *S. aureus* strains, with a molecular weight of 26,000 Da and a pI between 7.0 and 7.5 [48, 140]. An increase in the secretion of SEl-K was obtained when coexpressed with SEB (K-2014). However, regardless of the variation in SEl-K secretion amount *in vitro*, similar levels of SEl-K accumu‐ lation were found *in vivo* [141]. SEl-K was commonly detected in clinical isolates (more than half) and almost all USA300 strains. In addition, a genetic variation of *sel-k* was discovered, with 6 variants found among 20 clinical isolates [141].

#### *2.2.7. Staphylococcal Enterotoxin-Like Toxin Type L (sel-l and SEl-L)*

First noticed on *egc* from *S. aureus* strain A900322 [142], *sel-l* was identified on pathogenicity island SaPIbov (15,891 bp) from a bovine mastitis *S. aureus* isolate RF122 (*sel-l*) in 2001, with a molecular weight of 26,000 Da and an isoelectric point of 8.5 [105]. Lacking emetic activity, SEl-L was found to exhibit a number of biological properties similar to other SEs, including superantigenicity, pyrogenicity, enhancement of endotoxin shock, and lethality in rabbits when administered via subcutaneous mini-osmotic pumps, but the protein lacked emetic activity [105].

#### *2.2.8. Staphylococcal Enterotoxin-Like Toxin Type M (sel-m and SEl-M)*

In 2001, *sel-m* was reported to be located on the *egc* (enterotoxigenic gene cluster) together with *sel-g*, *sel-I*, *sel-k*, and *sel-l*, and SEl-M was found to exhibit superantigenicity activity with specific Vβ pattern [142]. However, the emetic activity has not been elucidated yet. Most clinical *S. aureus* strains harboring *egc* were found to carry such SEs regardless of the diseases, suggesting the potential derivation of SEs and the putative cluster of SE genes from *egc*.

#### *2.2.9. Staphylococcal Enterotoxin-Like Toxin Type N (sel-n and SEl-N)*

From the *egc* reported in 2001, *sel-n* was also found to be located between *sel-i* and *sel-g* [142]. A study was conducted on the cloning and expression of *sel-m* and *sel-n* from *S. aureus* strain FRI-1230, demonstrating that SEl-M and SEl-N were capable of stimulating T cells and inhibiting K562-ADM and B16 cells with an equivalent level to that of SEC2 [143]. Although superantigenicity had been verified, the emetic activity of SEl-N is still unclear [144].
