*2.2.10. Staphylococcal Enterotoxin-Like Toxin Type O (sel-o and SEl-O)*

In 2001, *sel-o* was identified from the *egc* cluster, on which other 4 SEs and 2 pseudogenes were also located, including *sel-i*, *sel-j*, *sel-m*, *sel-n*, *Ψent1*, and *Ψent2* [142]. However, the biological and biochemical properties of *sel-o* remains unclear despite validation of its superantigenicity [144].

#### *2.2.11. Staphylococcal Enterotoxin-Like Toxin Type P (sel-p and SEl-P)*

In 2001, *sel-p* (previously called *sep*) was first discovered from the genome of MRSA N315, and its biological properties were fully characterized in 2005 (with *sel-p* from *S. aureus* strain Sagal isolated from an SE unidentified food poisoning outbreak in Japan), including superantige‐ nicity (induction of a substantial proliferative response and production of cytokines) and emetic activity (at relatively high dose as 50–150 μg/animal) [123, 145]. According to this study, SEl-P was detected in 60% of the 30 *sel-p*-positive *S. aureus* isolates, and all 10 strains harboring *seb* and *sel-p* produced SEB but not SEl-P, suggesting that inactivation of the *sel-p* locus associates with a particular SE genetic constitution [123]. Most recently, colonization with *selp*-positive MRSA increased the risk of bacteremia, which indicated *sel-p* as a predictive virulence factor for invasive disease [146].

#### *2.2.12. Staphylococcal Enterotoxin-Like Toxin Type Q (sel-q and SEl-Q)*

In 2002, a member of the new subfamily (group V), *sel-q* (from *S. aureus* strain MN NJ) was identified and located directly 5 of *sel-k*, with a molecular weight of 26,000 Da and isoelectric point between 7.5 and 8.0 [147]. Despite a lack of emetic activity (incapability in neither lethality in rabbits nor emetic activity in monkeys), *sel-q* had been found to possess superantigenicity, pyrogenicity, and ability to enhance endotoxin shock.

#### *2.2.13. Staphylococcal Enterotoxin-Like Toxin Type R (sel-r and SEl-R)*

In 2003, *sel-r* was recovered and identified from 4 *S. aureus* strains (Fukuoka 5, Fukuoka 6, Fukuoka 7, and Fukuoka 8) isolated from patients with nausea, vomiting, and diarrhea from a food poisoning outbreak occurred at a lunch-box shop in Fukuoka prefecture of Japan in September 1997 [148]. Located on 2 types of plasmid, pBI485 (and pBI485-like plasmids, encoding *sed* and *sel-j* as well) and pK0311 (pF5, pF6, and pF7), *sel-r* was found to most closely related to *sel-g* [148]. Investigation on the biological properties of SEl-R revealed its superan‐ tigenicity (T-cell stimulation activity via MHC-II) and emetic activity (induction of a reaction in animals within 5 h at 100 μg/kg) [148–150]. SEl-R production was also verified in seropositive *S. aureus* strains [148, 149]. A survey was conducted on the SEl-R production from 24 *sed*positive *S. aureus* isolates, and *sel-r* expression was detectable from 22 isolates despite carriage of variant *sed* gene for seven strains lacking SED production [151].

#### *2.2.14. Staphylococcal Enterotoxin-Like Toxin Type S (sel-s and SEl-S)*

Two novel SE-like genes, *sel-s* and *sel-t*, had been reported on the plasmid pF5, where *sel-j* and *sel-r* were located. SEl-S (rSES) was characterized for biological properties, including super‐ antigenicity (specific stimulation of human T cells via MHC-II APC) and emetic activity (induction of emetic reactions in monkeys) [150].

#### *2.2.15. Staphylococcal Enterotoxin-Like Toxin Type T (sel-t and SEl-T)*

As aforementioned, a first identification of *sel-t* was reported on plasmid pF5 harbored by *S. aureus* strain Fukuoka 5 from SFP. Similar to SEl-S, SEl-T was found to exhibit both superan‐ tigenicity and emetic activity (induction of a delayed reaction after 24 h or 5 days postadmi‐ nistration). Data from the emetic study on SEs involved in the SFP outbreak in Fukuoka in 1997 combined with emesis studies in house musk shrews (similar as in the monkeys) suggest that SEl-R and SEl-S were validated to be the causative toxins of vomiting [150].

### *2.2.16. Staphylococcal Enterotoxin-Like Toxin Type U (sel-u and SEl-U)*

From sequencing of 24 *S. aureus* strains harboring *egc*, *sel-u* was identified on 4 of the tested strains [152]. SEl-U was found to result from sequence divergence in the *Ψent1* and *Ψent2* pseudogenes, as *sel-u* was located between *sel-iv* and *sel-n* in *egc* of strain 382F (AY158703) with replacement of the *Ψent1* and *Ψent2* between *sel-iv* and *sel-n* in *egc* of strain Mu50 (AP003363) [144, 152]. A variant *sel-u*, designated as *sel-u2*, was recovered from an atypical *egc* locus and generated by a limited deletion in the pseudogenes *Ψent1* and *Ψent2*, which contained superantigenicity for activation of T-cell families Vβ-13.2 and Vβ-14 [144].

#### *2.2.17. Staphylococcal Enterotoxin-Like Toxin Type V (sel-v and SEl-V)*

In a broad surveillance on *egc* from 666 clinical *S. aureus* isolates, 63% [421/666] strains were positive for *egc* locus [144]. The archetypal *egc* harboring 5 SEs and 2 pseudogenes was found in 409 strains, and a novel SE-like toxin, designated as *sel-v*, was discovered from an atypical *egc* locus from *S. aureus* strain A900624 [144]. SEl-V was generated by recombination between *sel-m* and *sel-i*, and its superantigenicity for activation of T-cell families Vβ-6, Vβ-18 and Vβ-21 has also been validated.

#### *2.2.18. Staphylococcal Enterotoxin-Like Toxin Type X (sel-x and SEl-X)*

In 2011, *sel-x* was discovered from the core genome of 95% of phylogenetically diverse *S. aureus* strains with human and animal origins, including 17 distinct allelic variants (*sel-x*1 to *sel-x*14, *sel-xov*, *sel-xbov*1, and *sel-xbov*2). Acquisition of *sel-x* includes the horizontal transfer by a *S. aureus* progenitor, allelic diversification by point mutation, and assortative recombination, which explains the high genetic diversity of *sel-x*. With a unique predicted structure, SEl-X was well characterized by biological activities, including superantigenicity (activation of Vβspecific T cells), pyrogenicity, and endotoxin enhancement. It is also noteworthy that SEl-X produced by strain USA300 (CA-MRSA) had been found to be responsible for the lethality in a rabbit model, which suggested a novel virulence determinant of CA-MRSA disease patho‐ genesis.

#### **2.3. Pathogenicity Islands (PAIs) and** *S. aureus* **PIs (SaPIs)**

#### *2.3.1. PAIs*

In 1986, before the first report of PAIs by Hacker et al. in Werner Goebel's group of Germany in 1994, two large segments had been found to be capable of deletion and thus enable the host bacterial to produce hemolysin and loss of P. fimbriae [139, 153, 154]. Considered to be foreign DNA segments integrated into the bacterial genome, such segments existed within pathogenic isolates (cause of virulence) but not on highly genetically similar nonpathogenic strains [153]. As a subclass of genomic islands, PAIs are defined as a group of gene clusters encoding bacterial virulence on a large DNA segment (ranging from 20 to l00 kb) located on the bacterial chromosome [139, 154–156]. PAIs are acquired by microorganisms via horizontal gene transfer via transduction, conjugation, and transformation. Acquisition of PAIs may rapidly and radically alter the genome of a bacterium, consequently strengthening or reducing its fitness within the host [154, 157]. Pathogens are capable of harboring one or more PAIs associated with one or more virulence genes. PAIs are capable of encoding genetic products, including secretory proteins (such as type III secretion system), cell surface proteins (such as erythrocy‐ tolysin, fimbriae, and heme binding factors), signal transduction systems, and regulation systems [139, 155–157].

As distinct DNA regions are present in the genome of pathogenic bacteria and absent in nonpathogenic strains (despite same or close species), typical PAIs are composed of mobility genes (such as integrases) commonly located at the beginning of the island and close to the tRNA locus or the respective attachment site. A number of virulence genes (V1–V4) are frequently interspersed with other mobility elements including insertion sequence (IS) elements (Isc, complete insertion element) or remnants of IS elements (ISd, defective insertion element) [153, 155, 156]. Commonly flanked by direct repeats (DR) and IS elements, PAIs are often genetically unstable and comprise some potential mobile components, such as IS elements, integrase, transposase, and plasmid replication initiation sites. As DNA sequences ranging from 16 to 20 bp (with maximum of 130 bp) with sequence repetition, DR plays a critical role in insertion and deletion (as recognition sites), such as integration of bacterio‐ phages. Although combination of IS elements may be capable of mediating transfer of large DNA fragments, insertion mediated by IS also leads to inactivation of genes as well [153, 154, 156]. Consequently, PAIs are capable of deletion with distinct frequencies and loss of virulence traits encoded by PAIs are reported to occur at higher frequency than that encoded by mutation. PAIs are commonly inserted in the backbone genome of the host strain, typically located to specific sites such as tRNA loci or adjacent to tRNA genes, or sites associated with plasmid and phage integration, due to highly conserved genes encoding tRNAs among various bacterial species [153, 154].

PAIs differ from host chromosomes in GC content and codon usage, which may account for the discovery of novel PAIs and maintenance of the divergent nucleotide composition from the horizontally acquired DNA. Based on significant differences with respect to bacterial virulence, GC content, and codon usage, a hypothesis was proposed that such characteristics may be bestowed from DNA segments on the plasmid and phage; thus, acquisition of PAIs and the emergence of new pathogenic organisms can be correlated [153, 156].

#### *2.3.2. SaPIs*

As mobile pathogenicity islands with length ranging from 14 to 17 kb and carriage of genes for superantigen toxins and other virulence factors, SaPIs have been responsible for the TSS and other superantigen-related diseases, especially SE-like toxins. Located in specific loci of the chromosome and induced by bacteriophages, SaPIs are capable of incorporating small infective phage-like particles via a program of excision-replication-packaging. Containing most of the staphylococcal toxins and virulence factors, SaPIs facilitate the horizontal acquis‐ ition of MGEs and, thus, play an important role in the evolution of *Staphylococcus* [139, 154, 155].

#### *2.3.2.1. SaPI families*

Aside from SCC*mec* (introduction in detail as below), a large number of toxin genes have been found in SaPIs, including SE-like toxins and TSST. Several types of SaPIs have been identified. SaPI1 was found to be inserted in an *attC* site close to the *tyrB* gene and flanked by the region of *tst* gene, with a length of 15,233 bp [139]. The characteristic features of SaPI1 include mobility and instability, whereas SaPI2 was identified as a second locus. Transduction between SaPI1 and SaPI2 by via helper phage was demonstrated, and stable integration of these 2 SaPIs without phages had also been verified [139, 155, 156]. Inserted at the 3 end of the GMP synthase gene, SaPIbov was identified in a bovine isolate of *S. aureus* by PAI related to SaPI1, with a length of 15,891 bp and carriage of *sec*, *sel-l*, and *tst*. SaPI3 was identified to contain *sel-k* and *sel-q* [157]. SaPIs with similar structure between SaPI3 and SaPI1 had also been reported.

#### *2.3.2.2. νSa families*

Up to date, 7 conservative PAI types had been discovered in *S. aureus*, namely, νSa1 (including SaPI1 and SaPI3), νSa2 (SaPIbov), νSa3, νSa4 (including SaPI2), νSaα, νSaβ, and νSaγ [139, 155–157]. νSa1 to νSa4 were found to contain integrase genes as putative elements of genetic mobility. Derivation of *int* and *att* sites from phage genome was pointed out, as both were found in SaPIs. On the contrary, νSaα and νSaβ harbor transposase genes, which may be derived form transposons. Comprising SaPI1 and SaPI3, the νSa1 locus of CA-MRSA also carries a large number of genes encoding enterotoxins and TSST. Similarly, the loci of both νSa2 from CA-MRSA and SaPIbov were found to contain enterotoxins and TSST. Capable of high-frequency deletion and formation of an episomal circular DNA, νSa3 was identified in CA-MRSA MW2 and Mu50, and one type of νSa3 harbors novel allelic forms of *sec* and *sel-l*. With a lower frequency of excision than that of νSa3, the νSa4 family contains several allelic forms of a genomic island, and type I νSa4 carries *sec* and *sel-l* [157]. Despite the presence of both νSaα and νSaβ in all sequenced *S. aureus* genomes, the size and number of ORFs in νSaα, as well as the size and gene composition of νSaβ, were found to be highly variable, with neither SaPIs spontaneously excised from the chromosome [145]. The composition of νSaα includes 11 allelic forms of *set* genes (encoding exotoxins), *lukDE* genes (encoding leukotoxins), and lipoprotein gene clusters. However, all varieties of νSaβ contain a gene cluster for serine proteins and superantigen genes absent in CA-MRSA but present in HA-MRSA strains. Adjacent to short DRs, the locus *etd* PAI contains exfoliative toxins *etd* and *edin-B* (encoding exfoliative toxins), IS element, and restriction/modification system [158].

#### *2.3.2.3. Development and evolution of pathogenicity*

The mechanisms of horizontal gene transfer in prokaryotic cells include transduction, conju‐ gation, and transformation, among which phage transduction has been the primary transmis‐ sion drivers of genes among different species and thus plays an important role in the formation of PAI [159]. The formation of PAIs may include 5 stages [160] as follows: (i) acquisition of virulence gene via horizontal gene transfer regulated by an operon and derived from "gene pool" of varied environment; (ii) integration of foreign genes (commonly derived from various complex genes of different donors) into the bacterial chromosome or plasmid via site-specific recombination or other mechanisms, following similar rules to complete integration and shaping obvious structure of genetic island; (iii) evolvement of MGEs into PAI via restructure, gene elimination, and acquisition of other genetic materials, during which the gene compo‐ nents associated with mobility may be inactivation or lost, such as origin of plasmid replication, self-transmissible plasmid tra and phage int; (iv) induced expression of foreign genes under temperate environment; and (v) acquisition or elimination of genetic information constantly via a serious of recombination, insertion or elimination, by which PAI may retrieve MGE and obtain the ability of excision and transfer the whole PAI from chromosome to another recipient bacterium.

Evolution of the bacterial genome may significantly influence its pathogenicity, mainly including point mutations, recombination, and horizontal gene transfer. Despite slow evolu‐ tion due to relatively low frequency of point mutation, the horizontal gene transfer of large genetic segments (such as PAIs and SaPIs) undoubtedly speeds up the exchange of bacterial genes (as "quantum leap" in short time), leading to the consequent appearance and spread of various novel mutations or variants [161, 162].
