Kunihiro Shinagawa

Detection of seg, seh, and sei genes in Staphylococcus aureus Isolates and Determination of the Enterotoxin Productivities of S. aureus Isolates Harboring seg, seh, or sei Genes

ABSTRACT To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%) S. aureus isolates were found to be positive for one or more se genes. The se genotype was classified into 14 genotypes. seg and sei coexisted in the same S. aureus strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most of the S. aureus isolates harboring seg and about 60% of the isolates harboring sei did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.

Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids.

ABSTRACT We identified and characterized a novel staphylococcal enterotoxin-like putative toxin, which is named SER. Nucleotide sequencing analysis of the ser gene revealed that ser was most closely related to the seg gene. The ser gene product, SER, was successfully expressed as a recombinant protein in an Escherichia coli expression system, and recombinant SER (rSER) showed significant T-cell stimulation activity. The SER production in ser-harboring Staphylococcus aureus strains was confirmed by Western blot analysis using anti-rSER antibody. Moreover, ser was seen to be encoded by at least two types of plasmids. In particular, one kind of plasmid encoding the ser gene has been known as a sed- and sej-carrying pIB485-related plasmid.

Identification and Characterization of Two Novel Staphylococcal Enterotoxins, Types S and T

ABSTRACT In addition to two known staphylococcal enterotoxin-like genes (selj and selr), two novel genes coding for two superantigens, staphylococcal enterotoxins S and T (SES and SET), were identified in plasmid pF5, which is harbored by food poisoning-related Staphylococcus aureus strain Fukuoka 5. This strain was implicated in a food poisoning incident in Fukuoka City, Japan, in 1997. Recombinant SES (rSES) specifically stimulated human T cells in a T-cell receptor Vβ9- and Vβ16-specific manner in the presence of major histocompatibility complex (MHC) class II+ antigen-presenting cells (APC). rSET also stimulated T cells in the presence of MHC class II+ APC, although its Vβ skewing was not found in reactive T cells. Subsequently, we examined the emetic activity of SES and SET. We also studied SElR to determine emetic activity in primates. This toxin was identified in previous studies but was not examined in terms of possession of emetic activity for primates. rSES induced emetic reactions in two of four monkeys at a dose of 100 μg/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 μg/kg, and in the other one, the reaction occurred 5 days postadministration. rSElR induced a reaction in two of six animals within 5 h at 100 μg/kg. On this basis, we speculate that the causative toxins of vomiting in the Fukuoka case are SES and SER. Additionally, SES, SER, and SET also induced emesis in house musk shrews as in the monkeys.

Characterization of Novel Staphylococcal Enterotoxin-Like Toxin Type P

ABSTRACT We investigated the biological properties of a novel staphylococcal enterotoxin (SE)-like toxin type P (SElP). SElP induced a substantial proliferative response and the production of cytokines interleukin-2, gamma interferon, tumor necrosis factor alpha, and interleukin-4 from human T cells when administered at a concentration of 0.4 pM (0.01 ng/ml) or more. The expression of major histocompatibility complex class II molecules on accessory cells was required for T-cell stimulation by SElP. SElP selectively stimulated a vast number of human T cells bearing receptors Vβ 5.1, 6, 8, 16, 18, and 21.3. These results indicated that SElP acts as a superantigen. SElP proved to be emetic in the house musk shrew emetic assay, although at a relatively high dose (50 to 150 μg/animal). A quantitative assay of SElP production with 30 Staphylococcus aureus strains harboring selp showed that 60% of these strains produced significant amounts of SElP in vitro. All 10 strains carrying seb and selp produced SEB but not SElP, suggesting the inactivation of the selp locus in S. aureus strains with a particular se gene constitution.

Analytical methods for Bacillus cereus and other Bacillus species.

Bacillus cereus can give rise to two distinct forms of foodborne disease, the emetic and the diarrhoeal syndromes. The emetic syndrome is believed to be associated with an emetic toxin pre-formed in food. Cooked rice is the most common vehicle, and the symptoms are similar to those of Staphylococcus aureus intoxication. The diarrhoeal type is caused by an enterotoxin and the symptoms generally parallel those of the Clostridium perfringens food poisoning. The heat resistance of B. cereus spores and the non-fastidious nature of the organism facilitates its survival and/or growth in a wide variety of foods. This review describes analytical methods available for the isolation, identification, and enumeration of the organism, in addition to details about biological and immunological methods for toxin assay. Data are also presented concerning the incidence and epidemiology of B. cereus food poisoning around the world, and especially in Japan.

Vaccination with Nontoxic Mutant Toxic Shock Syndrome Toxin 1 Protects against Staphylococcus aureus Infection

To investigate whether vaccination with nontoxic mutant toxic shock syndrome toxin 1 (mTSST-1) can protect against Staphylococcus aureus infection, mice were vaccinated with mTSST-1 and challenged with viable S. aureus. Survival in the mTSST-1-vaccinated group was higher, and bacterial counts in organs were significantly lower than those of control mice. Passive transfer of mTSST-1-specific antibodies also provided protection against S. aureus-induced septic death. Interferon (IFN)-gamma production in the serum samples and spleens from vaccinated mice was significantly decreased compared with that in controls, whereas interleukin-10 titers were significantly higher in vaccinated mice. IFN-gamma and tumor necrosis factor-alpha production in vitro were significantly inhibited by serum samples from mTSST-1-immunized mice but not from control mice. These results suggest that vaccination with mTSST-1 devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibodies as well as by the down-regulation of IFN-gamma production.

Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable bacterial superantigenic toxins causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and its emetic signal pathway. We investigated a mechanism of SEA‐induced emesis using a small emetic animal model, house musk shrew. SEA‐induced emesis in the animals was inhibited by a 5‐hydroxytryptamine (5‐HT) synthesis inhibitor and a 5‐HT3 receptor antagonist. SEA could increase 5‐HT release in the small intestine. Pre‐treatment with 5,7‐dihydroxytryptamine (5,7‐DHT) markedly inhibited SEA‐induced emesis. SEA‐induced emesis was also abolished by surgical vagotomy. Furthermore, cannabinoid (CB) receptor agonists inhibited SEA‐induced emesis, and the action was reversed by a CB1 antagonist. Both 5‐HT release and CB1 receptor expression were found in the mucosal and myenteric plexus of the intestine. Moreover, a CB1 receptor agonist significantly decreased the 5‐HT release in the intestine. These results demonstrate that SEA induces 5‐HT release in intestine, rather than in brain, and that the 5‐HT3 receptors on vagal afferent neurons are essential for SEA‐stimulated emesis. In addition, SEA‐induced emesis is downregulated by the CB system through decreasing 5‐HT release in intestine.

Induction of Emetic Response to Staphylococcal Enterotoxins in the House Musk Shrew (Suncus murinus)

ABSTRACT The emetic responses induced by staphylococcal enterotoxin A (SEA), SEB, SEC2, SED, SEE, SEG, SEH, and SEI in the house musk shrew (Suncus murinus) were investigated. SEA, SEE, and SEI showed higher emetic activity in the house musk shrew than the other SEs. SEB, SEC2, SED, SEG, and SEH also induced emetic responses in this animal model but relatively high doses were required. The house musk shrew appears to be a valuable model for studying the mechanisms of emetic reactions caused by SEs.

Comparative prevalence of superantigenic toxin genes in meticillin-resistant and meticillin-susceptible Staphylococcus aureus isolates.

A total of 118 meticillin-resistant Staphylococcus aureus (MRSA) and 140 meticillin-susceptible S. aureus (MSSA) isolates from different patients in the same time period were comprehensively searched using a multiplex PCR for the classical and recently described superantigenic toxin gene family comprising the staphylococcal enterotoxin genes sea to ser and the toxic shock syndrome toxin 1 gene, tst-1. Both MRSA and MSSA isolates carried a number of superantigenic toxin genes, but the MRSA isolates harboured more superantigenic toxin genes than the MSSA isolates. The most frequent genotype of the MRSA isolates was sec, sell and tst-1 together with the gene combination seg, sei, selm, seln and selo, which was found strictly in combination in 69.5% of the isolates tested. In contrast, possession of the sec, sell and tst-1 genes in MSSA isolates was significantly less than in MRSA (2.1 vs 77.1%, respectively), although they also often contained the combination genes (25.0%). This notable higher prevalence in MRSA isolates indicated that possession of the sec, sell and tst-1 genes in particular appeared to be a habitual feature of MRSA. Moreover, these were mainly due to the fixed combinations of the mobile genetic elements type I nuSa4 encoding sec, sell and tst-1, and type I nuSabeta encoding seg, sei, selm, seln and selo. Analysis of the relationship between toxin genotypes and the toxin gene-encoding profiles of mobile genetic elements has a possible role in determining superantigenic toxin genotypes in S. aureus.

Biological Properties of Staphylococcal Enterotoxin-Like Toxin Type R

ABSTRACT We investigated the biological properties of a novel staphylococcal enterotoxin-like putative toxin, staphylococcal enterotoxin-like toxin type R (SElR). Major histocompatibility complex class II molecules were required for T-cell stimulation by SElR. SElR stimulated T cells bearing receptors Vβ 3, 11, 12, 13.2, and 14. These results suggested that SElR acts as a superantigen.