Distribution, polymorphism and temporal expression of egc in Staphylococcus aureus isolates from various foods in China

Abstract

Staphylococcal food poisoning (SFP) is characterized by diarrhea and vomiting resulting from the ingestion of staphylococcal enterotoxins (SEs) contaminated foods. The genome-located enterotoxin gene cluster (egc) encodes superantigens performed different clinical severities than traditional SEs. In this study, we identified 336 Staphylococcus aureus isolates from various foods in China and investigated the presences of egc, 5 classical SE genes, accessory gene regulator (agr) and sarA. Subsequently, the egc subtypes were assayed and the mRNA expression of egc and relative genes in the cell cycle were analyzed in selected isolates. As a result, egc was more prevalent than all traditional SE genes in foodborne S. aureus isolates, and egc1 was the predominant subtype. During the growth cycle, the expression patterns of egc and those of traditional SE genes were similar in tested isolates: the enterotoxin mRNAs peaked at the post-exponential growth phase and then rapidly decreased. Simultaneously, the agr system was activated and the sarA expression was enhanced. However, ZJY58, the only selected isolate which did not harbor agr, performed significantly lower peak of egc expression than other isolates. Hence, these data of gene typing and expression described a general profile of egc in food-derived S. aureus, and would have potential use in the control of SFP.

Introduction

Staphylococcal food poisoning (SFP), resulting from the ingestion of staphylococcal enterotoxins (SEs) in food, is one of the most common food-borne diseases worldwide (Marrack & Kappler, 1990). SEA is the first discovered SE serological type in the 1960's, and to date, twenty-one SEs and enterotoxin-like superantigens (SEls) (SEA – SEE, SEG – SEI, SElJ – SElQ, SER – SET, SElU – SElV) have been identified (Omoe et al., 2005; Ono et al., 2008; Thomas et al., 2006; Zhang, Iandolo, & Stewart, 1998). SEs and SEls all possess superantigenic activity which leads to massive T-cell proliferation and cytokine release, whereas only SEs have been proven to be emetic (Marrack & Kappler, 1990). These toxins are produced by enterotoxigenic strains of coagulase-positive staphylococci (mainly Staphylococcus aureus) in food with high protein content (Lina et al., 2004).

The distribution of SE genes in enterotoxigenic S. aureus strains is different. All of the SE genes are located on mobile genetic elements such as phages and plasmids, except an operon termed the (egc) in a genomic pathogenicity island (Jarraud et al., 2001). egc was discovered by Monday and Bohach (2001) and Jarraud et al. (2001), comprising five genes and two pseudogenes designated selo, selm, sei, ψent1, ψent2, seln and seg (egc1). Subsequently, Letertre, Perelle, Dilasser, and Fach (2003) reported two egc polymorphisms, egc2 and egc3, with sequence divergences in the ψent1–ψent2 pseudogenes and minor variants for the egc-encoded SE genes. Thomas et al. (2006) identified a further genetic variation of the egc locus in strain A900624, which generated two new genes designated selv and selu2. Consequently, at least 4 different egc subtypes were suggested: (i) egc1 (harboring seo, sem, sei, ψent1, ψent2, seln and seg), in strain A900322 (GenBank ID: AF285760), (ii) egc2 (containing seu instead of ψent1 and ψent2), in strain FRI137 (GenBank ID: AY205306), (iii) egc3 (containing sei, seu, sen, and seg variants), in strain 382F (GenBank ID: AY158703), (iv) egc4 (containing selo, selv, selu2, seln and seg), in strain A900624 (GenBank ID: EF030428) (Collery, Smyth, Tumilty, Twohig, & Smyth, 2009).

Epidemiological studies show that different patterns of SE genes are associated with different clinical severities. Although egc-encoded genes are the most prevalent SE genes in food and clinical-borne S. aureus isolates (Becker, Friedrich, Peters, & Eiff, 2004; Fueyo, Mendoza, Alvarez, & Martin, 2005) and considered to be the “nursery” of staphylococcal superantigens (Jarraud et al., 2001), they are rarely identified in toxic shock cases (Ferry et al., 2005; Proft & Fraser, 2003). In fact, egc-encoded SEs are more frequently identified in foodborne strains than in invasive isolates, and their presence is negatively correlated with severity of S. aureus sepsis (Ferry et al., 2005). However, the biological mechanisms of the difference between egc and non-egc superantigens are not clear, and moreover, conflicts were shown in the scanty reports. Dauwalder and coworkers described that a non-egc SE (SEA) is more potent than an egc-encoded SE (SEG) in inducing proinflammatory and Th1 response in human PBMC (Dauwalder et al., 2009; Holtfreter et al., 2004). However, Grumann and coworkers reported very similar aspects of immune cell activation and downstream gene expression caused by egc and non-egc SEs, and presumed that the distinct biological behavior of egc and non-egc superantigens is due to their differential release by S. aureus (Grumann et al., 2008).

The pathogenesis of S. aureus depends on a tremendous range of adaptive or accessory genes. Accessory gene regulator (agr) is one of the key global quorum-sensing systems of S. aureus, regulating a large set of virulence genes at the level of post-transcription and translation (Novick, 2003). RNAIII, the effector molecule of the staphylococcal agr, are activated by the auto-inducing peptides and functions by interacting with other transcription factors such as sarA (Chien, Manna, & Cheung, 1998). Some staphylococcal superantigens (SEB, SEC and SED) are positively regulated by agr (Gaskill & Khan, 1988; Vojtov, Ross, & Novick, 2002; Zhang & Stewart, 2000), and others (SEA) are stable when agr is activated (Tremaine, Brockman, & Betley, 1993).

However, as the most prevalent SEs gene in S. aureus, egc has not been investigated for its association with agr system and relative regulators. Further, only few literatures have reported the egc polymorphism in foodborne S. aureus (Blaiotta, Fusco, Eiff, Villani, & Becker, 2006) and the temporal expression of egc during the bacterial growth (Derzelle, Dilasser, Duquenne, & Deperrois, 2009). Here, we characterized egc and classical SE genes in 336 S. aureus isolates from various kinds of Chinese foods, and identified the egc polymorphism. Then, the mRNA expressions of egc during the cell growth were assayed in selected isolates.