Next Article in Journal
Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats
Previous Article in Journal
Molecular Biology of Escherichia coli Shiga Toxins’ Effects on Mammalian Cells
Open AccessArticle

Prevalence and Genetic Diversity of Staphylococcal Enterotoxin (-Like) Genes sey, selw, selx, selz, sel26 and sel27 in Community-Acquired Methicillin-Resistant Staphylococcus aureus

1
Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan
2
Sapporo Clinical Laboratory, Inc., Sapporo 060-0005, Japan
*
Author to whom correspondence should be addressed.
Toxins 2020, 12(5), 347; https://doi.org/10.3390/toxins12050347
Received: 11 April 2020 / Revised: 20 May 2020 / Accepted: 21 May 2020 / Published: 23 May 2020
(This article belongs to the Section Bacterial Toxins)

Abstract

Staphylococcal enterotoxins (SEs) are virulence factors of Staphylococcus aureus associated with various toxic diseases due to their emetic and superantigenic activities. Although at least 27 SE(-like) genes have been identified in S. aureus to date, the newly identified SE(-like) genes have not yet been well characterized by their epidemiological features. In this study, the prevalence and genetic diversity of SE gene sey and SE-like genes selw, selx, selz, sel26, and sel27 were investigated for 624 clinical isolates of community-acquired methicillin-resistant S. aureus (CA-MRSA). The most prevalent SE(-like) gene was selw (92.9%), followed by selx (85.6%), sey (35.4%) and selz (5.6%), while sel26 and sel27 were not detected. Phylogenetically, sey, selw, selx, and selz were discriminated into 7, 10, 16, and 9 subtypes (groups), respectively. Among these subtypes, sey was the most conserved and showed the highest sequence identity (>98.8%), followed by selz and selx. The SE-like gene selw was the most divergent, and four out of ten genetic groups contained pseudogenes that may encode truncated product. Individual subtypes of SE(-like) genes were generally found in isolates with specific genotypes/lineages of S. aureus. This study revealed the putative ubiquity of selw and selx and the prevalence of sey and selz in some specific lineages (e.g., ST121) in CA-MRSA, suggesting a potential role of these newly described SEs(-like) in pathogenicity.
Keywords: Staphylococcus aureus; enterotoxin; sey; selw; selx; selz Staphylococcus aureus; enterotoxin; sey; selw; selx; selz

1. Introduction

Staphylococcus aureus is one of the most common pathogens in humans and is responsible for various diseases ranging from skin and soft tissue infections to severe and often deadly infections such as bacteremia [1]. Clinical isolates of S. aureus have been distinguished between methicillin-susceptible and -resistant S. aureus (MSSA and MRSA, respectively) based on the presence of the mecA gene associated with resistance to beta-lactam antibiotics. While healthcare-associated MRSA (HA-MRSA) was initially recognized as a major nosocomial pathogen worldwide, the emergence and spread of community-associated MRSA (CA-MRSA) since the 1990s has been a global public health concern until today [2].
A group of superantigens, i.e., staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), is produced by most clinical isolates of S. aureus as etiological factors of toxic diseases including food poisoning and toxic shock syndrome [3]. To date, at least 27 SE or SE-like proteins have been identified, and most of them (SEA-SEE, SEG-SEI, SEK-SET, SEY) were demonstrated to have emetic activity in animals [3,4,5]. SElJ is a non-emetic protein, as well as TSST-1, while the emetogenicity of remaining SE-like proteins is yet to be determined. Bacterial superantigens that are produced by staphylococci and streptococci are phylogenetically classified into five groups, among which four groups (I, II, III, and V) include SEs and TSST-1 [3,4,6]. The prevalence of SE genes including sea-see and seg-selu in S. aureus has been analyzed in many studies of isolates from bacteremia [7,8], diabetic foot ulcers [9], cystic fibrosis [10], and colonization in healthy humans [11,12], as well as those from animals and the environment [13,14,15]. Although the distribution of SE genes sea-see and seg-seo (or -seu) to clinical isolates of HA- and CA-MRSA was also investigated previously [16,17,18], the prevalence of more recently described SE(-like) genes (sey, selw, selx, selz, sel26, and sel27) has not yet been well characterized.
sey was first described as a SET-like gene having 32% amino acid sequence identity to SET, and phylogenetically related to the group I superantigen including the TSST-1 gene [19]. This gene was identified in isolates from food poisoning, skin diseases, nasal colonization, and bovine mastitis, and its recombinant protein was proved to have superantigen activity in human mononuclear cells and emetic activity in a primate animal [19,20]. selx, which is classified as a group I superantigen, was revealed to be present at a high rate in the core genome of genetically diverse S. aureus strains [21,22]. In addition to superantigenic activity, SELX has an ability to bind to neutrophils, which inhibits its phagocytosis function, and thus is presumed to be implicated in the virulence of CA-MRSA [23]. selz, which belongs to SEB group (group II), was reported in the RF122 strain from bovine mastitis [4]. sel26 and sel27 (GenBank accession no. MF370874) were reported in S. aureus, S. argenteus and S. schweizeri and assigned into SEI and SEB groups (group V and II), respectively [5].
selw was reported as a novel SE-like gene by Okumura et al. [24] in the S. aureus strain N315 (GenBank accession no. BA000018, locus_tag SA1430) based on the nomenclature standard of SE [25]. It exhibited similarity to sea (36% amino acid sequence identity) and was classified into the same phylogenetic group as SEA (group III) [24]. selw had been previously used to refer to selu2 [26,27,28,29], an allelic variant of selu; both selu variants are phylogenetically distinct from the gene described by Okumura et al. [4,30,31]. Thus, selw has been discriminated from selu2 [4,8,31,32]. In the present study, selw denotes the SE-like gene that was described for the N315 strain [24].
We previously analyzed 624 CA-MRSA clinical isolates that were derived from outpatients in Hokkaido, Northern main island of Japan, for their molecular epidemiological and genetic characteristics [33], and reported the predominance of SCCmec IIa MRSA, and also the presence of SCCmec IVa-ST8 isolates (USA300 clone) carrying Panton–Valentine leukocidine (PVL) genes and ST5/ST764 MRSA-harboring arginine catabolic mobile element (ACME). Furthermore, we identified ST8 MRSA as having SCCmec IVl, which had been designated “CA-MRSA-J” and presumably emerged in Japan and other regions of Asia. In the present study, the prevalence of the newly described SE(-like) genes (sey, selw, selx, selz, sel26, and sel27) in these CA-MRSA isolates were investigated and their genetic diversity was analyzed phylogenetically.

2. Results

2.1. The Prevalence of sey, selw, selx, selz, sel26, and sel27

The prevalence of the SE(-like) genes among 624 CA-MRSA isolates is summarized in Table 1 and SE(-like) gene profiles in different sequence types (STs) of the selected 100 isolates are shown in Table 2. The most prevalent SE(-like) gene was selw (92.9%), followed by selx (85.6%), sey (35.4%), and selz (5.6%), while no isolates harbored sel26 and sel27. selw was commonly detected in isolates with genotypes coa-IIa-ST5/ST764 (98.9%), coa-VIIa-ST1 (90.2%), and coa-IIIa-ST8 (86.5%), and also found in coa-Va-ST121, coa-Ia-ST89, and coa-VIIb-ST45 isolates. spa types t002, t1784, t008 were the most common in coa-IIa-ST5/764, coa-VIIa-ST1, and coa-IIIa-ST8, respectively. While sey showed a high prevalence in coa-IIa-ST5/764 and coa-Va-ST121, this gene was less frequently detected in coa-IIIa-ST8 and coa-VIIa-ST1 (30–40%). selx was prevalent in coa-Va-ST121, coa-VIIa-ST1, coa-IIa-ST5/764, and coa-IIIa-ST8 with a detection rate of more than 80%. selz was identified at a high rate in only coa-Va-ST121 and coa-Ia-ST89 isolates.
The profiles of SE(-like) genes were generally unique to the STs of isolates (Table 2). Panton–Valentine Leukocidin/arginine catabolic mobile element (PVL/ACME)-positive ST8 (SCCmecIVa-t008, USA300 clones) isolates had only four SE(-like) genes (sek, seq, selw and selx), while ST1, ST5, ST764 isolates harbored more genes with high rates of selw and selx. PVL/ACME-negative ST8 isolates of the CA-MRSA/J clone had sec, sel, and sep, in addition to selw and selx, while non-CA-MRSA/J ST8 isolates exhibited different profiles of SE(-like) genes.
Co-detection of selw-selx-sey-selz was found in ST121 and ST89 MRSA isolates, while selw-selx-sey was found in ST5 and its SLV (ST764 and ST5425), ST8 (PVL+/ACME+, non-CA-MRSA/J), and ST45 isolates. Though sey, selw, selx, and selz are not located in an enterotoxin gene cluster (egc) in the chromosome of S. aureus [28,30], egc-2 (seg-sei-sem-sen-seo-seu) was co-detected with sey/selw/selx/selz in ST5 and ST764 (CC5), and also in ST121. egc-1 (seg-sei-sem-sen-seo) was found in ST45 and ST5425 isolates, together with selw and selx.
sey and selz were more commonly identified in SCCmec V MRSA than in SCCmec II and III isolates. No distinct difference was found in the prevalence of selw and selx depending on SCCmec types. Detection rates of the SE(-like) genes analyzed in the present study were generally similar among the different specimens from which MRSA isolates were derived.

2.2. Phylogenetic and Sequence Analysis of sey, selw, selx, and selz

For the 149 selected isolates belonging to different coa genotypes, nucleotide sequences of full-length ORF of sey, selw, selx, and selz were determined (44, 24, 67, and 14 isolates, respectively). Phylogenetic trees of these genes were constructed by the maximum likelihood method for the SE(-like) genes analyzed in the present study together with sequences in the GenBank database for representative S. aureus strains and those representing subtypes of individual SE(-like) genes (Figure 1).
SE gene sey was genetically differentiated into at least seven subtypes (sey1–sey7), including three variants, (sey1–sey3) described by Aziz et al. [20] (Figure 1a, Figure S1). The nucleotide sequence identity among the seven sey subtypes was more than 98.8% (Table S1). Phylogenetically, sey1 and sey4, and sey2 and sey7 were assigned into a same group and sey5 was genetically close to sey3, having only one nucleotide (amino acid) difference. sey sequences of the CA-MRSA isolates were mostly assigned into sey5, which included various genotypes, i.e., coa-Ia-ST89, coa-IIa-ST5/ST764, coa-IIIa/ST8, and coa-VIIa-ST1/ST12. The second most common subtype was sey1, which was identified in coa-Va-ST121 and coa-IIa-ST5 isolates.
selw had been classified into six groups (1–6) in our previous study on colonizing S. aureus isolates from food handlers [11]. In the present study, ten selw groups (group 1–10), including six groups previously reported, were discriminated, and group 1 was subdivided into four clusters (1a–1d) (Figure 1b, Figure S2). selw sequences were highly divergent, exhibiting >93% identity among all the groups, and 95–98% within group 1 (Table S2). Truncated products deduced from selw sequences were identified in isolates of groups 4, 8, and 9, and RF122 strain (group 1c). These were caused by internal stop codons, resulting in a lack of 85–130 amino acids at the C-terminal portion, while intact SElW consists of 250 amino acids (Figure S2). selw of the CA-MRSA isolates was assigned into groups 1 (1a,b), 2, 3, 4, 7, and 8, which contained isolates with coa-Ia-ST89/coa-VIIb-ST45, coa-IIIa/VIIa-ST1/ST8, coa-VIIa-ST1, coa-IIa-ST5 (CC5), coa-Va-ST121, and coa-IVa-ST30, respectively.
Wilson et al. classified selx into at least 14 alleles (subtypes) [21] having a sequence identity of >94% (Table S3). According to this classification, most of the selx in the CA-MRSA isolates were assigned into selx1, selx2, selx5, and selx10, which contained coa-IIa/VIIa-ST1/ST5/ST764, coa-IIIa-ST8, coa-VIIa-ST1, and coa-Va-ST121 isolates, respectively (Figure 1c, Figure S3). Only isolate SC533 was assigned into a new subtype, selx16. Alignment of SElX amino acid sequences revealed that a sialic acid-binding region consisting of 16 amino acids [22,23] is conserved among all of the subtypes, except for a single position (Figure S3).
selz sequences determined in the present study and those obtained from the GenBank database were classified into nine subtypes (selz1–selz9), among which selz1–selz5 were phylogenetically assigned to a single group (selz1 group) (Figure 1d, Figure S4). Nucleotide sequence identity among different selz subtypes was 96–99% (Table S4). selz of the CA-MRSA was assigned to selz1 and selz6 groups, which were identified in coa-Va-ST121/coa-IIa-ST5/764 and coa-Ia-ST89 isolates, respectively. selz of S. argenteus and S. aureus were classified into the same group (selz1 and selz6 groups), although S. argenteus clusters of sey, selw, and selx were distinct from that of S. aureus (Figure 1a–d).

3. Discussion

In this study, we investigated the prevalence of six SE(-like) genes (sey, selw, selx, selz, sel26, and sel27) in CA-MRSA clinical isolates and revealed a high prevalence of selw and selx, a lower prevalence of sey and selz, and an absence of sel26 and sel27. It was notable that these six genes are distributed also to S. argenteus, although the prevalence of other SE(-like) genes was very low, except for sec and the enterotoxin gene cluster (egc, seg-sei-sem-sen-seo) in some isolates [34]. The prevalence of SE(-like) genes analyzed in the present study was different among S. argenteus depending on lineages: sey, sel26 and sel27 in ST2250; selx in ST2198, and selw in ST1223, while selz was found in all three STs [34]. Phylogenetically, sey, selw, and selx of S. argenteus formed a distinct cluster from those of S. aureus. In contrast, selz was not genetically differentiated by the two staphylococcal species. These findings suggest that sey, selw, selx, and selz might have been archaic virulence determinants in S. aureus complex (SAC) and passed on to progeny through their deletion and genetic evolution in individual lineages of S. aureus and S. argenteus. Although the presence of sel26 and sel27 was reported in ST27 and ST772 S. aureus strains in addition to ST2250 S. argenteus strains [5], our present study suggests that these genes may not play an important role in the virulence of CA-MRSA.
A high prevalence of selx was reported for S. aureus clinical isolates from blood, diabetic foot ulcers, and cystic fibrosis, as well as for those from colonization [8,9,10,11], showing comparable or higher detection rates than those of sea, sec, and egc. Furthermore, selx was revealed to be highly conserved, despite the presence of various subtypes. Therefore, it is suggested that selx may be involved in any universal role in the virulence of S. aureus, which is presumably associated with superantigenic activity as well as neutrophil-binding activity [21,23]. Though SELlX was initially reported as a novel virulence factor for the USA300 clone [21], this SE-like protein may be implicated in pathogenesis of broad CA-MRSA clones. Although selx was differentiated into more than 16 subtypes, including newly assigned types in the present study, all the SElX subtypes have a conserved motif of sialic acid binding [22,23], which is essential for its superantigen- and neutrophil-binding activity. Thus, the biological function of SElX is suggested to be same among its subtypes.
In contrast, selw was revealed to be divergent and classified into ten genetic groups. Four selw groups contained putative pseudogenes that encode truncated products, which lose 30–50% amino acids of intact SElW. Thus, such truncated products are suggested to be dysfunctional or have reduced function compared with intact SElW. The highly divergent nature of selw may suggest that this gene is a remnant of archaic, universal virulence determinant in SAC, having a less significant role in virulence than selx.
SEY, with three subtypes (sey1–sey3), was reported as a novel SE as well as superantigen in S. aureus and identified in isolates from skin diseases with a detection rate of 17–22% [20]. In our present study for CA-MRSA, a higher prevalence (35.4%) was noted, particularly in coa-I-III, V and VII. In addition to the three subtypes described previously, a new subtype sey5, which is genetically related to sey3, was identified and contained in a higher number of isolates belonging to various genotypes (coa-Ia/IIa/IIIa/VIIa-ST1/5/8/12/89/764) than sey1 (coa-IIa/Va-ST5/121). Thus, the sey variant group comprising sey5 and sey3 may be more prevalent among CA-MRSA, presumably associated with its virulence. Aziz et al. [20] reported that the effects of SEY on human lymphocytes were slightly different among SEY variants (SEY1, SEY2, SEY3), but not significantly. Because the additional SEY subtypes (SEY4-7) detected in our study were genetically close to all of SEY1-3 and the divergent amino acid positions were also similar, the newly identified SEY subtypes probably have the same function as that reported for SEY1-3.
It was notable in this study that ST121 isolates (coa-Va) had sey, selw, selx, and selz, leading to the highest detection rates of sey and selz in coa-Va. Because the ST121 S. aureus clone is characterized by various virulence factors and referred to as “hypervirulent” [35], sey, selw, selx, and selz are also suggested to be involved in increased virulence in this clone. The putative ubiquity of selw and selx and the prevalence of sey and selz in some specific lineages (e.g., ST121) in CA-MRSA may provide the possibility for their potential pathogenic role in human infections.
CA-MRSA has been reported to possess mostly SCCmec IV or V in clones represented by ST1, ST8, ST30, ST59, and ST80 [2]. However, in our previous studies in northern Japan, SCCmec II was predominant among CA-MRSA, accounting for more than 72%, while remaining MRSA had SCCmec IV or V [33,36]. SCCmec II-MRSA with ST5, which is known as the “New York/Japan clone, has been one of the typical HA-MRSA clones and detected predominantly in our previous studies [37,38]. Thus, our present results may represent the situation of the SE(-like) gene in an area where SCCmec II is dominant among CA-MRSA. Further study may be necessary to reveal the prevalence of the newer SE genes among CA-MRSA with type IV and V SCCmec.
This study revealed the ubiquitous distribution of selw and selx in CA-MRSA and the prevalence of sey and selz dependent on some specific lineages. These findings suggest a potential role of these newly described SE(-like) in the pathogenicity of CA-MRSA.

4. Materials and Methods

4.1. CA-MRSA Isolates

A total of 624 non-duplicate CA-MRSA isolates, which had been analyzed for their molecular epidemiological characteristics (SCCmec, ST, coa-type, etc.) in our previous study [33], were used as the subject of the present study. These isolates were derived from clinical specimens of outpatients who visited hospitals and clinics in the prefecture of Hokkaido, Japan, during a nine-month period (July 2013 and March 2014). Coagulase genotype (coa) is the genetic classification of the staphylocoagulase gene based on its diversity in N-terminal divergent regions (D1, D2) and is discriminated by the multilex PCR scheme [39]. The most prevalent coagulase (coa) genotype was IIa (455, 72.9%), followed by IIIa (74, 11.9%), VIIa (51, 8.2%), Ia (17, 2.7%), and Va (16, 2.6%). The dominant SCCmec type was IIa (439, 70.4%) followed by IVa (78, 12.5%), V (34, 5.4%) IVl (24, 3.8%), and IVh (13, 2.1%) [33].

4.2. Genetic Analysis of S. aureus (CA-MRSA) Isolates

SE(-like) genes sey, selw, selx, selz, sel26, and sel27 were detected by uniplex PCR, using primers listed in Table S5 The nucleotide sequences of the full-length ORF of SE(-like) genes were determined by direct sequencing with PCR products, using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an automated DNA sequencer (ABI PRISM 3100). Primers were designed to amplify ORF of the SE(-like) genes in this study (Table S5). The phylogenetic dendrogram of toxin genes was constructed by the maximum likelihood method using the MEGA.6 software package. Multiple alignment of nucleotide/amino acid sequences determined in the present study and those retrieved from the GenBank database was performed by Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/), which was also used for the calculation of the sequence identity. In our previous study [33], the coa genotype and SCCmec type were determined for all the isolates, and spa, ST, and enterotoxin profiles were characterized for 31 isolates. In the present study, spa type and ST were determined for more representative 118 and 69 isolates, respectively, as described previously [33]. For the 69 isolates, of which ST was determined, the prevalence of all the SE(-like) genes was analyzed [38,40].

4.3. GenBank Accession Numbers

The nucleotide sequences of sey, selw, selx, selz were deposited in the GenBank database under the accession numbers listed in Table S6.

Supplementary Materials

The following are available online at https://www.mdpi.com/2072-6651/12/5/347/s1, Figure S1: Alignment of amino acid and nucleotide sequences of SEY, Figure S2: Alignment of SElW amino acid sequences of all the MRSA isolates analyzed in the present study, and representative strains of all the selw phylogenetic groups, Figure S3: Alignment of SElX amino acid sequences representing subtypes SElX1-SElX16, Figure S4: Alignment of SElZ amino acid sequences representing subtypes SElZ1-SElZ9, Tables S1–S4: Nucleotide Sequence identities (percentage) of sey, selw, selx, and selz gene among selected S. arueus isolates and those of reported S. argenteus strain, respectively, Table S5: Primers used in the present study, Table S6: GenBank accession numbers assigned to sey, selx, selw and selz sequences determined in the present study.

Author Contributions

Conceptualization, M.S.A. and N.K.; methodology, M.S.A. and N.K.; investigation, M.S.A., N.U., and M.K.; resources, M.I. and S.H.; data curation, N.K.; writing—original draft preparation, M.S.A.; writing—review and editing, N.K.; supervision, N.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant No. 18K10054.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Lowy, F.D. Staphylococcus aureus infections. N. Engl. J. Med. 1998, 339, 520–532. [Google Scholar] [CrossRef] [PubMed]
  2. David, M.Z.; Daum, R.S. Community-associated methicillin-resistant Staphylococcus aureus: Epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev. 2010, 23, 616–687. [Google Scholar] [CrossRef] [PubMed]
  3. Spaulding, A.R.; Salgado-Pabón, W.; Kohler, P.L.; Horswill, A.R.; Leung, D.Y.; Schlievert, P.M. Staphylococcal and streptococcal superantigen exotoxins. Clin. Microbiol. Rev. 2013, 26, 422–447. [Google Scholar] [CrossRef] [PubMed]
  4. Wilson, G.J.; Tuffs, S.W.; Wee, B.A.; Seo, K.S.; Park, N.; Connelley, T.; Guinane, C.M.; Morrison, W.I.; Fitzgerald, J.R. Bovine Staphylococcus aureus Superantigens Stimulate the Entire T Cell Repertoire of Cattle. Infect Immun. 2018, 86, e00505–e00518. [Google Scholar] [CrossRef] [PubMed]
  5. Zhang, D.F.; Yang, X.Y.; Zhang, J.; Qin, X.; Huang, X.; Cui, Y.; Zhou, M.; Shi, C.; French, N.P.; Shi, X. Identification and characterization of two novel superantigens among Staphylococcus aureus complex. Int. J. Med. Microbiol. 2018, 308, 438–446. [Google Scholar] [CrossRef] [PubMed]
  6. Xu, S.X.; McCormick, J.K. Staphylococcal superantigens in colonization and disease. Front. Cell. Infect. Microbiol. 2012, 2, 52. [Google Scholar] [CrossRef]
  7. Holtfreter, S.; Grumann, D.; Schmudde, M.; Nguyen, H.T.; Eichler, P.; Strommenger, B.; Kopron, K.; Kolata, J.; Giedrys-Kalemba, S.; Steinmetz, I.; et al. Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates. J. Clin. Microbiol. 2007, 45, 2669–2680. [Google Scholar] [CrossRef]
  8. Roetzer, A.; Haller, G.; Beyerly, J.; Geier, C.B.; Wolf, H.M.; Gruener, C.S.; Model, N.; Eibl, M.M. Genotypic and phenotypic analysis of clinical isolates of Staphylococcus aureus revealed production patterns and hemolytic potentials unlinked to gene profiles and source. BMC Microbiol. 2016, 16, 13. [Google Scholar] [CrossRef]
  9. Vu, B.G.; Stach, C.S.; Salgado-Pabón, W.; Diekema, D.J.; Gardner, S.E.; Schlievert, P.M. Superantigens of Staphylococcus aureus from patients with diabetic foot ulcers. J. Infect. Dis. 2014, 210, 1920–1927. [Google Scholar] [CrossRef]
  10. Fischer, A.J.; Kilgore, S.H.; Singh, S.B.; Allen, P.D.; Hansen, A.R.; Limoli, D.H.; Schlievert, P.M. High Prevalence of Staphylococcus aureus Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates. Genes (Basel) 2019, 10, 1036. [Google Scholar] [CrossRef]
  11. Aung, M.S.; San, T.; Aye, M.M.; Mya, S.; Maw, W.W.; Zan, K.N.; Htut, W.H.W.; Kawaguchiya, M.; Urushibara, N.; Kobayashi, N. Prevalence and Genetic Characteristics of Staphylococcus aureus and Staphylococcus argenteus Isolates Harboring Panton-Valentine Leukocidin, Enterotoxins, and TSST-1 Genes from Food Handlers in Myanmar. Toxins (Basel) 2017, 9, 241. [Google Scholar] [CrossRef] [PubMed]
  12. Fooladvand, S.; Sarmadian, H.; Habibi, D.; van Belkum, A.; Ghaznavi-Rad, E. High prevalence of methicillin resistant and enterotoxin gene-positive Staphylococcus aureus among nasally colonized food handlers in central Iran. Eur. J. Clin. Microbiol. Infect. Dis. 2019, 38, 87–92. [Google Scholar] [CrossRef] [PubMed]
  13. Chao, G.; Bao, G.; Cao, Y.; Yan, W.; Wang, Y.; Zhang, X.; Zhou, L.; Wu, Y. Prevalence and diversity of enterotoxin genes with genetic background of Staphylococcus aureus isolates from different origins in China. Int. J. Food Microbiol. 2015, 211, 142–147. [Google Scholar] [CrossRef] [PubMed]
  14. Mello, P.L.; Moraes Riboli, D.F.; Pinheiro, L.; de Almeida Martins, L.; Vasconcelos Paiva Brito, M.A.; Ribeiro de Souza da Cunha Mde, L. Detection of Enterotoxigenic Potential and Determination of Clonal Profile in Staphylococcus aureus and Coagulase-Negative Staphylococci Isolated from Bovine Subclinical Mastitis in Different Brazilian States. Toxins (Basel) 2016, 8, 104. [Google Scholar] [CrossRef]
  15. Shen, M.; Li, Y.; Zhang, L.; Dai, S.; Wang, J.; Li, Y.; Zhang, L.; Huang, J. Staphylococcus enterotoxin profile of China isolates and the superantigenicity of some novel enterotoxins. Arch. Microbiol. 2017, 199, 723–736. [Google Scholar] [CrossRef]
  16. Naimi, T.S.; LeDell, K.H.; Como-Sabetti, K.; Borchardt, S.M.; Boxrud, D.J.; Etienne, J.; Johnson, S.K.; Vandenesch, F.; Fridkin, S.; O’Boyle, C.; et al. Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus infection. JAMA 2003, 290, 2976–2984. [Google Scholar] [CrossRef]
  17. Hu, D.L.; Omoe, K.; Inoue, F.; Kasai, T.; Yasujima, M.; Shinagawa, K.; Nakane, A. Comparative prevalence of superantigenic toxin genes in meticillin-resistant and meticillin-susceptible Staphylococcus aureus isolates. J. Med. Microbiol. 2008, 57, 1106–1112. [Google Scholar] [CrossRef]
  18. Shukla, S.K.; Karow, M.E.; Brady, J.M.; Stemper, M.E.; Kislow, J.; Moore, N.; Wroblewski, K.; Chyou, P.H.; Warshauer, D.M.; Reed, K.D.; et al. Virulence genes and genotypic associations in nasal carriage, community-associated methicillin-susceptible and methicillin-resistant USA400 Staphylococcus aureus isolates. J. Clin. Microbiol. 2010, 48, 3582–3592. [Google Scholar] [CrossRef]
  19. Ono, H.K.; Sato’o, Y.; Narita, K.; Naito, I.; Hirose, S.; Hisatsune, J.; Asano, K.; Hu, D.L.; Omoe, K.; Sugai, M.; et al. Identification and Characterization of a Novel Staphylococcal Emetic Toxin. Appl. Environ. Microbiol. 2015, 81, 7034–7040. [Google Scholar] [CrossRef]
  20. Aziz, F.; Hisatsune, J.; Yu, L.; Kajimura, J.; Sato’o, Y.; Ono, H.K.; Masuda, K.; Yamaoka, M.; Salasia, S.I.O.; Nakane, A.; et al. Staphylococcus aureus Isolated from Skin from Atopic-Dermatitis Patients Produces Staphylococcal Enterotoxin Y, Which Predominantly Induces T-Cell Receptor Vα-Specific Expansion of T Cells. Infect. Immun. 2020, 88, e00360–e00419. [Google Scholar] [CrossRef]
  21. Wilson, G.J.; Seo, K.S.; Cartwright, R.A.; Connelley, T.; Chuang-Smith, O.N.; Merriman, J.A.; Guinane, C.M.; Park, J.Y.; Bohach, G.A.; Schlievert, P.M.; et al. A novel core genome-encoded superantigen contributes to lethality of community-associated MRSA necrotizing pneumonia. PLoS Pathog. 2011, 7, e1002271. [Google Scholar] [CrossRef] [PubMed]
  22. Langley, R.J.; Ting, Y.T.; Clow, F.; Young, P.G.; Radcliff, F.J.; Choi, J.M.; Sequeira, R.P.; Holtfreter, S.; Baker, H.; Fraser, J.D. Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors. PLoS Pathog. 2017, 13, e1006549. [Google Scholar] [CrossRef] [PubMed]
  23. Tuffs, S.W.; James, D.B.A.; Bestebroer, J.; Richards, A.C.; Goncheva, M.I.; O’Shea, M.; Wee, B.A.; Seo, K.S.; Schlievert, P.M.; Lengeling, A.; et al. The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function. PLoS Pathog. 2017, 13, e1006461. [Google Scholar] [CrossRef] [PubMed]
  24. Okumura, K.; Shimomura, Y.; Murayama, S.Y.; Yagi, J.; Ubukata, K.; Kirikae, T.; Miyoshi-Akiyama, T. Evolutionary paths of streptococcal and staphylococcal superantigens. BMC Genom. 2012, 13, 404. [Google Scholar] [CrossRef] [PubMed]
  25. Lina, G.; Bohach, G.A.; Nair, S.P.; Hiramatsu, K.; Jouvin-Marche, E.; Mariuzza, R. International Nomenclature Committee for Staphylococcal Superantigens. Standard nomenclature for the superantigens expressed by Staphylococcus. J. Infect. Dis. 2004, 189, 2334–2336. [Google Scholar] [CrossRef]
  26. Thomas, D.Y.; Jarraud, S.; Lemercier, B.; Cozon, G.; Echasserieau, K.; Etienne, J.; Gougeon, M.L.; Lina, G.; Vandenesch, F. Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster. Infect. Immun. 2006, 74, 4724–4734. [Google Scholar] [CrossRef]
  27. Collery, M.M.; Smyth, C.J. Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes ψent1 and ψent2 and the selu or seluv gene using PCR-RFLP. J. Med. Microbiol. 2007, 56, 208–216. [Google Scholar] [CrossRef]
  28. Argudín, M.Á.; Mendoza, M.C.; Rodicio, M.R. Food poisoning and Staphylococcus aureus enterotoxins. Toxins (Basel) 2010, 2, 1751–1773. [Google Scholar] [CrossRef]
  29. Fisher, E.L.; Otto, M.; Cheung, G.Y.C. Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning. Front. Microbiol. 2018, 9, 436. [Google Scholar] [CrossRef]
  30. Benkerroum, N. Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview. Crit. Rev. Food Sci. Nutr. 2018, 58, 1943–1970. [Google Scholar] [CrossRef]
  31. Tuffs, S.W.; Haeryfar, S.M.M.; McCormick, J.K. Manipulation of Innate and Adaptive Immunity by Staphylococcal Superantigens. Pathogens 2018, 7, 53. [Google Scholar] [CrossRef] [PubMed]
  32. Chieffi, D.; Fanelli, F.; Chob, G.S.; Schubert, J.; Blaiotta, G.; Franz, C.M.A.P.; Bania, J.; Fusco, V. Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk. Food Microbiol. 2020, 90, 103482. [Google Scholar] [CrossRef] [PubMed]
  33. Aung, M.S.; Kawaguchiya, M.; Urushibara, N.; Sumi, A.; Ito, M.; Kudo, K.; Morimoto, S.; Hosoya, S.; Kobayashi, N. Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Outpatients in Northern Japan: Increasing Tendency of ST5/ST764 MRSA-IIa with Arginine Catabolic Mobile Element. Microb. Drug. Resist. 2017, 23, 616–625. [Google Scholar] [CrossRef] [PubMed]
  34. Aung, M.S.; Urushibara, N.; Kawaguchiya, M.; Sumi, A.; Takahashi, S.; Ike, M.; Ito, M.; Habadera, S.; Kobayashi, N. Molecular Epidemiological Characterization of Staphylococcus argenteus Clinical Isolates in Japan: Identification of Three Clones (ST1223, ST2198, and ST2550) and a Novel Staphylocoagulase Genotype XV. Microorganisms 2019, 7, 389. [Google Scholar] [CrossRef] [PubMed]
  35. Rao, Q.; Shang, W.; Hu, X.; Rao, X. Staphylococcus aureus ST121: A globally disseminated hypervirulent clone. J. Med. Microbiol. 2015, 64, 1462–1473. [Google Scholar] [CrossRef]
  36. Kawaguchiya, M.; Urushibara, N.; Kuwahara, O.; Ito, M.; Mise, K.; Kobayashi, N. Molecular characteristics of community-acquired methicillin-resistant Staphylococcus aureus in Hokkaido, northern main island of Japan: Identification of sequence types 6 and 59 Panton-Valentine leucocidin-positive community-acquired methicillin-resistant Staphylococcus aureus. Microb. Drug Resist. 2011, 17, 241–250. [Google Scholar]
  37. Kawaguchiya, M.; Urushibara, N.; Yamamoto, D.; Yamashita, T.; Shinagawa, M.; Watanabe, N.; Kobayashi, N. Characterization of PVL/ACME-positive methicillin-resistant Staphylococcus aureus (genotypes ST8-MRSA-IV and ST5-MRSA-II) isolated from a university hospital in Japan. Microb. Drug Resist. 2013, 19, 48–56. [Google Scholar] [CrossRef]
  38. Aung, M.S.; Urushibara, N.; Kawaguchiya, M.; Sumi, A.; Shinagawa, M.; Takahashi, S.; Kobayashi, N. Clonal Diversity and Genetic Characteristics of Methicillin-Resistant Staphylococcus aureus Isolates from a Tertiary Care Hospital in Japan. Microb. Drug Resist. 2019, 25, 1164–1175. [Google Scholar] [CrossRef]
  39. Hirose, M.; Kobayashi, N.; Ghosh, S.; Paul, S.K.; Shen, T.; Urushibara, N.; Kawaguchiya, M.; Shinagawa, M.; Watanabe, N. Identification of staphylocoagulase genotypes I-X and discrimination of type IV and V subtypes by multiplex PCR assay for clinical isolates of Staphylococcus aureus. Jpn. J. Infect. Dis. 2010, 63, 257–263. [Google Scholar]
  40. Aung, M.S.; Urushibara, N.; Kawaguchiya, M.; Aung, T.S.; Mya, S.; San, T.; New, K.M.; Kobayashi, N. Virulence factors and genetic characteristics of methicillin-resistant and -susceptible Staphylococcus aureus isolates in Myanmar. Microb. Drug Resist. 2011, 17, 525–535. [Google Scholar] [CrossRef]
Figure 1. Phylogenetic dendrogram of sey (a), selw (b), selx (c), selz (d) constructed by the maximum likelihood method using MEGA6. The tree was statistically supported by bootstrapping with 1000 replicates, and genetic distances were calculated by the Kimura two-parameter model. The variation scale is provided at the bottom. The percentage bootstrap support is indicated by the values at each node (values <80 are omitted). The isolates analyzed in the present study are shown with closed circles. The S. argenteus cluster in sey, selw, and selx is shown at the bottom ((a), (b), (c)), while selz of the S. argenteus strain is indicated by a diamond (d). Subtypes/groups of individual SE(-like) genes are shown by boxes on the right. Closed triangles with selw groups and a strain in (b) represent genes encoding truncated proteins. Genotypes of isolates or GenBank accession numbers are shown in parenthesis followed by strain names.
Figure 1. Phylogenetic dendrogram of sey (a), selw (b), selx (c), selz (d) constructed by the maximum likelihood method using MEGA6. The tree was statistically supported by bootstrapping with 1000 replicates, and genetic distances were calculated by the Kimura two-parameter model. The variation scale is provided at the bottom. The percentage bootstrap support is indicated by the values at each node (values <80 are omitted). The isolates analyzed in the present study are shown with closed circles. The S. argenteus cluster in sey, selw, and selx is shown at the bottom ((a), (b), (c)), while selz of the S. argenteus strain is indicated by a diamond (d). Subtypes/groups of individual SE(-like) genes are shown by boxes on the right. Closed triangles with selw groups and a strain in (b) represent genes encoding truncated proteins. Genotypes of isolates or GenBank accession numbers are shown in parenthesis followed by strain names.
Toxins 12 00347 g001aToxins 12 00347 g001bToxins 12 00347 g001cToxins 12 00347 g001d
Table 1. The prevalence of sey, selw, selx and selz among 624 CA-MRSA isolates with different genotypes, SCCmec types and origins.
Table 1. The prevalence of sey, selw, selx and selz among 624 CA-MRSA isolates with different genotypes, SCCmec types and origins.
GenotypeTotal No. of IsolatesNo. of Isolates With SE(-Like) Gene *1 (%)
coa Genotypespa Type (n = 149) *2seyselwselxselz
Iat375 (3)178 (47.1)8 (47.1)13 (76.5)12 (70.6)
IIat002 (56), t548 (2), t2487 (2), t001 (1), t045 (2)455157 (34.5)450 (98.9)397 (87.3)5 (1.1)
IIIat008 (9), t4133 (2), t1767 (24), t5071 (1), t1627 (2), t1581(2)7422 (29.7)64 (86.5)60 (81.1)0
IVat019 (1)301 (33.3)00
Vat5110 (6), t10641 (10)1613 (81.3)8 (50)16 (100)14 (87.5)
VbNT (1)101 (100)1 (100)0
VIaND40000
VIIat1784 (23)5120 (39.2)46 (90.2)46 (90.2)4 (7.8)
VIIbt370 (2)31 (33.3)2 (66.7)1 (33.3)0
Total 624221 (35.4)580 (92.9)534 (85.6)35 (5.6)
SCCmec type
SCCmec I 201 (50)2 (100)0
SCCmec II 452154 (34.1)431 (95.4)393 (86.9)5 (1.1)
SCCmec IV 12541 (32.8)115 (92)106 (84.8)4 (3.2)
SCCmec V 3426 (76.5)30 (88.2)30 (88.2)26 (76.5)
SCCmec NT 1103 (27.3)3 (27.3)0
Specimen
sputum 13645 (33.1)130 (95.6)121 (89)9 (6.6)
urine 12940 (31)118 (91.5)102 (79.1)3 (2.3)
ear discharge 7635 (46.1)72 (94.7)70 (92.1)6 (7.9)
nasal discharge 7528 (37.3)73 (97.3)67 (89.3)7 (9.3)
pus 5724 (42.1)54 (94.7)52 (91.2)3 (5.3)
wound swab 2913 (44.8)27 (93.1)27 (93.1)3 (10.3)
eye swab 2912 (41.4)28 (96.6)26 (89.7)2 (6.9)
stool 339 (27.3)26 (78.8)24 (72.7)0
skin 2610 (38.5)24 (92.3)23 (88.5)2 (7.7)
Others *3 345 (14.7)28 (82.4)22 (64.7)0
NT, non-typable. *1 sel26 and sel27 were negative for all the isolates. *2 spa type and ST were determined for a total of 149 isolates comprising coa-Ia (3), coa-IIa (63), coa-IIIa (40), coa-IVa (1), coa-Va (16), coa-Vb (1) coa-VIIa (23), and coa-VIIb (2). ND, spa-typing not done. *3 Others included specimens of blood, bronchial lavage fluid, pharynx, aspirate, pleural fluid, joint fluid, HVS, catheter tip, drainage fluid, suction tube.
Table 2. The presence of enterotoxin(-like) genes in CA-MRSA isolates with different STs.
Table 2. The presence of enterotoxin(-like) genes in CA-MRSA isolates with different STs.
PVL/ACME GenesST (CC)Total No. of Isolates (n = 100)SE(-Like) Genes Identified *2
PVL+/ACME+ ST8 (CC8)9 *1sek, seq, selw, selx
PVL+/ACME- ST30 (CC30)1 *1sem, sen, seo, seu, selw, selx
ST59 (CC59)1 *1seb, sek, seq, selw, selx
PVL-/ACME+ST5/ST764 (CC5)15 *1seb (67%), sec (20%), seg, sei, sem, sen, seo, seu, sep (33%), selw, selx, sey
PVL-/ACME-ST8 (CC8) (CA-MRSA/J *3)5 *1sec, sel, sep, selw, selx
ST8 (CC8)14selj (29%), ser (29%), selw (93%), selx (93%), sey (43%)
ST5/ST764 (CC5)20seb (60%), sec (10%), seg, sei, sem, sen, seo, seu, selw, selx, sey (30%), selz (25%)
ST5425 (CC5)1seg, sei, sem, sen, seo, selw, selx
ST45 (CC45)2seg, sei, sem, sen, seo, selw, selx, sey
ST1 (CC1)13sea, sek, seq, selx (92%), selw (92%), sey (31%), selz (15%)
ST89 (CC89)8sem, seo, seu, selw, selx, sey, selz
ST121 (CC121)10seg, sei, sem, sen, seo, seu, selw (50%), selx, sey (80%), selz (85%)
ST12 (CC12)1sep, selw, selx, selz
ST, sequence type; CC, clonal complex. *1 For these 31 isolates, genotypes and enterotoxin genes profile had been already reported in our previous study [33]. *2 None of isolate had sed, see, ses and set. When SE(-like) genes were not present in all the isolates of the same ST, their detection rate (%) are indicated in parentheses. *3 CA-MRSA/J represents ST8 MRSA carrying SCCmec IVl.
Back to TopTop