Emetic potentials of newly identified staphylococcal enterotoxin-like toxins.

Staphylococcal enterotoxins (SEs) are a common causative agent of food poisoning. Recently, many new SE-like (SEl) toxins have been reported, although the role of SEls in food poisoning remains unclear. In this study, the emetic potentials of SElK, SElL, SElM, SElN, SElO, SElP, and SElQ were assessed using a monkey-feeding assay. All the SEls that were tested induced emetic reactions in monkeys at a dose of 100 µg/kg, although the numbers of affected monkeys were significantly smaller than the numbers that were affected after consuming SEA or SEB. This result suggests that these new SEs may play some role in staphylococcal food poisoning.

LC-MS analysis of the emetic toxin, cereulide, produced by Bacillus cereus.

A quantitative and chemical assay of cereulide produced in the cultures by some strains of Bacillus cereus was performed on a HPLC and a ESI electrospray ion trap mass analyzer, using the synthetic cereulide as a standard. All 20 strains of emetic B. cereus were found to produce 27 - 740 ng/ml of cereulide by the LC-MS analysis. In contrast, none of the 10 diarrheal strains produced it. 10(2) cfu/ml of the cereulide producible strain with a 210 ng/ml yield was inoculated into the 10% suspensions of 14 food products, and was incubated at 32°C for 24h. The B. cereus counts in the cultures grew in the order of 10(8) to 10(9) cfu/ml, although the bacteria could not grow in fruits, and the yields of cereulide ranged from 5.18µg in curry to 0.03µg/g of raw material and/or powder material, except for fruits. These culture supernatants were also tested for the biological activity in the HEp-2 cell culture assay. Consequently, a certain correlation was shown between the yields of cereulide and the HEp-2 vacuolation activities. In addition, the supernatants were administered i.p. to 5 Suncus marinus test animals. The emetic dose was calculated to be approximately 16µg/kg.

Submucosal mast cells in the gastrointestinal tract are a target of staphylococcal enterotoxin type A.

Staphylococcal enterotoxin A (SEA) is a leading causative toxin of staphylococcal food poisoning. However, it remains unclear how this toxin induces emesis in humans, primates, and certain experimental animals. To understand the mechanism of SEA-induced emesis, we investigated the behavior of SEA in the gastrointestinal (GI) tract in vivo using the house musk shrew (Suncus murinus). Immunofluorescence of GI sections showed that perorally administered SEA translocated from the lumen to the interior tissues of the GI tract and rapidly accumulated in certain submucosa cells. These SEA-binding cells in the submucosa were both tryptase- and FcεRIα-positive, suggesting these SEA-binding cells were mast cells. These SEA-binding mast cells were 5-hydroxytryptamine (5-HT)-positive, but the intensity of the 5-HT signal decreased over time compared to that of mast cells in the negative control. Furthermore, toluidine blue staining showed the number of metachromatic mast cells was decreased in the duodenal submucosa, suggesting that SEA binding induced degranulation and release of 5-HT from submucosal mast cells. These observations suggest that the target cells of SEA are submucosal mast cells in the GI tract and that 5-HT released from submucosal mast cells plays an important role in SEA-induced emesis.

High affinity of interaction between superantigen and T cell receptor Vbeta molecules induces a high level and prolonged expansion of superantigen-reactive CD4+ T cells.

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vß3(+)CD4(+) T cells exhibited a high level of expansion whereas the Vß11(+)CD4(+) T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vß11(+)CD4(+) T cells exhibited a high level of expansion while the Vß3(+)CD4(+) T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vß molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vß3(+)CD4(+) and Vß11(+)CD4(+) T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vß3/Vß11 ß chains and between SEA-MHC class II-molecule complex and Vß3(+)/Vß11(+) CD4(+) T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.

Interaction between superantigen and T-cell receptor Vbeta element determines levels of superantigen-dependent cell-mediated cytotoxicity of CD8(+) T cells in induction and effector phases.

Specific superantigens activate different T-cell fractions with distinct TCR V beta elements in association with MHC class II molecules and also induce SDCC against MHC class II(+) target cells. In the present study, to determine whether the responsiveness of each CD8(+) T-cell fraction expressing a different TCR V beta element is primarily determined by the TCR V beta, we compared the levels of proliferation and SDCC in V beta3(+) and V beta11(+) T cells upon stimulation with SEA. Upon stimulation with SEA(wt), the levels of proliferation were higher in V beta3(+) T cells than in V beta11(+) T cells. The levels of SDCC were also higher for the combination of V beta3(+) T cells and SEA(wt) than for the combination of V beta11(+) T cells and SEA(wt) during both the induction phase and the effector phase. In addition, upon stimulation with SEA(m), the levels of proliferation were higher in V beta11(+) T cells than in V beta3(+) T cells. And then, the levels of SDCC were also higher for the combination of V beta11(+) T cells and SEA(m) than for the combination of Vbeta3(+) T cells and SEA(m) during both the induction phase and the effector phase. These results suggest that the SAG-responsiveness of each CD8(+) T-cell fraction expressing a different TCR V beta element is primarily determined by the interaction between the TCR V beta element and the SAG.

Immunization with a nontoxic mutant of staphylococcal enterotoxin A, SEAD227A, protects against enterotoxin-induced emesis in house musk shrews.

BACKGROUND: Staphylococcal enterotoxins (SEs) are the most common cause of foodborne diseases and toxic shock throughout the world. However, no vaccine that prevents emesis induced by SEs has been described. METHODS: A nontoxic mutant of SEA, SEAD227A, was constructed by site-directed mutagenesis and was purified by means of the Escherichia coli expression system. House musk shrews, a small emetic animal model, were immunized with SEAD227A and then challenged with wild-type SEA. SEA-induced emesis was recorded for 3 h. Antibody production was analyzed by gel double-immunodiffusion assay. Neutralizing activities of the antibodies with respect to superantigenic and emetic activities were analyzed in vitro and in vivo. RESULTS: SEAD227A was devoid of both superantigenic and emetic activities but still retained its immunological activity. Immunization with SEAD227A strongly induced specific antibody production and provided significant protection against SEA-induced emesis. Antibodies from immunized shrews markedly inhibited the SEA-induced proliferation of spleen cells and also significantly ablated SEA-induced vomiting in the animals. CONCLUSIONS: These results suggest that vaccination with SEAD227A, which is devoid of toxic properties, provides protection against SEA-induced emesis. This nontoxic mutant and its specific antibodies might be useful in the prevention and treatment of staphylococcal food poisoning.

Identification and characterization of two novel staphylococcal enterotoxins, types S and T.

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 Vbeta9- and Vbeta16-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 Vbeta 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 microg/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 microg/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 microg/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.

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.

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-HT(3) 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-HT(3) 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.

Intranasal vaccination with a double mutant of staphylococcal enterotoxin C provides protection against Staphylococcus aureus infection.

Staphylococcus aureus expresses a repertoire of factors including staphylococcal exotoxins (SEs), exoenzymes, and numerous cell-associated components that contribute to the pathogenesis of disease. We constructed and expressed a nontoxic double mutant SEC (dmSEC), devoid of superantigenic activity, and investigated the ability of intranasal vaccination with dmSEC plus cholera toxin (CT) adjuvant to protect mice against S. aureus infection. Mice were vaccinated with dmSEC and inoculated with a viable S. aureus clinical isolate strain. The survival rate in the immunized mice was higher, and bacterial counts in the organs were significantly lower than those in the control group. Intranasal vaccination with dmSEC induced the production of SEC-specific antibodies such as IgG1, IgG2b and IgA. dmSEC-vaccinated mice elicited significantly higher titers of interleukin-4 (IL-4) and IL-10, and lower levels of interferon-gamma (IFN-gamma) after challenge with S. aureus compared with the control group. Furthermore, the sera from dmSEC-immunized mice significantly inhibited IFN-gamma and tumor necrosis factor-alpha production in vitro. These results indicate that intranasal vaccination with dmSEC devoid of superantigenic properties induces systemic immune responses and provides protection against S. aureus infection.

Characterization of novel staphylococcal enterotoxin-like toxin type P.

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 Vbeta 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 mug/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.

Staphylococcal enterotoxin A modulates intracellular Ca2+ signal pathway in human intestinal epithelial cells.

We demonstrate here that staphylococcal enterotoxin A (SEA) induces an increase in intracellular calcium ([Ca2+]i) in human intestinal epithelial cells and the [Ca2+]i is released from intracellular stores. SEA-induced increase of [Ca2+]i was clearly inhibited by treatment with a nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine and guanidine. Intestinal epithelial cells express endothelial NOS in resting cell condition, and express inducible NOS after stimulating with tumor necrosis factor (TNF)-alpha. TNF-alpha-pretreated cells showed a significant increase in [Ca2+]i that was also inhibited by the NOS inhibitor. These results suggest that SEA modulated [Ca2+]i signal is dependent on NOS expression in human intestinal epithelial cells.

Campylobacter jejuni isolated from retail poultry meat, bovine feces and bile, and human diarrheal samples in Japan: comparison of serotypes and genotypes.

To determine the significance of poultry and bovine as infectious sources of Campylobacter jejuni in Japan, the serotype distribution and pulsed-field gel electrophoresis (PFGE) patterns of poultry and bovine isolates were compared with those of isolates from patients with diarrhea in Akita (Japan). Serotypes O:2 and O:4-complex were common in human, poultry, and bovine isolates, and serotype O:23,36,53 was common in human and bovine isolates. SmaI PFGE patterns of isolates belonging to these serotypes were generated. Eight PFGE patterns were shared by poultry and human isolates and three patterns were shared by human and bovine isolates. Further analysis of the isolates having the same SmaI PFGE pattern by KpnI PFGE confirmed that four patterns and two patterns were still shared by poultry and human isolates, and bovine and human isolates, respectively. Thus, serotypic and genotypic data indicated a possible link between sporadic human campylobacteriosis and C. jejuni from retail poultry and bovine bile and feces, suggesting that bovine serves as an infectious source of C. jejuni in Japan, as is observed in other countries.

Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates.

We describe a comprehensive detection system for 18 kinds of classical and newly described staphylococcal superantigenic toxin genes using four sets of multiplex PCR. Superantigenic toxin genotyping of Staphylococcus aureus for 69 food poisoning isolates and 97 healthy human nasal swab isolates revealed 32 superantigenic toxin genotypes and showed that many S. aureus isolates harbored multiple toxin genes. Analysis of the relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic elements suggests its possible role in determining superantigenic toxin genotypes in S. aureus as combinations of toxin gene-encoding mobile genetic elements.

A mutant of staphylococcal enterotoxin C devoid of bacterial superantigenic activity elicits a Th2 immune response for protection against Staphylococcus aureus infection.

Staphylococcal enterotoxin C (SEC), a bacterial superantigenic exotoxin, is commonly produced by invasive Staphylococcus aureus isolates, especially methicillin-resistant strains and isolates from animal diseases. We constructed and expressed a nontoxic mutant SEC (mSEC) and investigated whether immunization with mSEC, which is devoid of superantigenic activity, can protect against S. aureus infection. Mice were immunized with mSEC and challenged with viable S. aureus. The bacterial counts in the organs of mSEC-immunized mice were significantly lower and the survival rate was higher than the corresponding values for the control group. Immunization with mSEC strongly induced the production of T-helper 2 type antibodies, immunoglobulin G1, and immunoglobulin G2b. The production of interleukin-10 (IL-10) and IL-4 was significantly greater in immunized mice challenged with S. aureus than in the control mice, whereas the production of gamma interferon (IFN-gamma) was significantly decreased in the immunized mice. The cytokine response in a spleen cell culture that was stimulated with heat-killed S. aureus or SEC showed that immunization with mSEC inhibited IFN-gamma production and up-regulated IL-10 production in vitro. Furthermore, IFN-gamma and tumor necrosis factor alpha production in vitro was significantly inhibited by sera from mSEC-immunized mice but not by sera from control mice. These results suggest that immunization with mSEC devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by SEC-specific neutralizing antibodies.

Comparative evaluation of the Desoxycholate Agar Nissui Food Stamp and swab methods for estimating coliform organisms in poultry processing plants after cleaning in Japan.

Bacterial control in poultry processing plants is very important, but the swab method for estimating bacterial contamination is somewhat troublesome in routine work. We compared the Desoxycholate Agar Nissui Food Stamp (DA-NFS) based on the agar contact method with the swab method to estimate coliform organisms from various equipments in four poultry processing plants after cleaning. Overall 104 surfaces for coliform organisms were evaluated. The results from 98 (94.2%) surfaces for coliform organisms were equivalent by the DA-NFS and swab methods and there were no significant differences between two methods (P > 0.05). The correlation coefficient between the DA-NFS and swab methods was 0.91. We conclude that the DA-NFS could be useful for routine coliform organisms examination in poultry processing plants after cleaning in Japan.

Biological properties of staphylococcal enterotoxin-like toxin type R.

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 Vbeta 3, 11, 12, 13.2, and 14. These results suggested that SElR acts as a superantigen.

Dose response for infection by Escherichia coli O157:H7 from outbreak data.

In 1996, an outbreak of E. coli O157:H7-associated illness occurred in an elementary school in Japan. This outbreak has been studied in unusual detail, making this an important case for quantitative risk assessment. The availability of stored samples of the contaminated food allowed reliable estimation of exposure to the pathogens. Collection of fecal samples allowed assessment of the numbers infected, including asymptomatic cases. Comparison to other published dose-response studies for E. coli O157:H7 show that the strain that caused the outbreak studied here must have been considerably more infectious. We use this well-documented incident as an example to demonstrate how such information on the response to a single dose can be used for dose-response assessment. In particular, we demonstrate how the high infectivity limits the uncertainty in the low-dose region.