Staphylococcal Enterotoxins A Research Articles

Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins

An electrochemiluminescent (ECL)-based multiplex immunoassay using Meso-Scale Discovery (MSD) technology was developed for detecting antibody response toward 10 Staphylococcus aureus (S. aureus) exotoxins. These 10 antigens included three different groups of toxins: 1) single component pore-forming toxins such as alpha- and delta-hemolysins, 2) the bicomponent pore-forming toxin Panton–Valentine leukocidin (PVL), comprised of LukS-PV and LukF-PV subunits, and 3) enterotoxin/superantigens — Staphylococcal enterotoxins A (SEA), B (SEB), C1 (SEC1), D (SED), K (SEK) and Toxic shock syndrome toxin-1 (TSST-1). Assay development included optimization steps with a conventional SEB ELISA-based serological assay and then optimized parameters were transferred and re-optimized in a singleplex ECL format. Finally, two pentaplex solid-phase ECL formats were developed. As proof of concept, one set of pentaplex ECL data was compared with conventional ELISA results. During the assay development controls were screened and developed for both the singleplex and multiplex assays. ECL-based multiplex assays were more sensitive with a wide dynamic range and proved more time-efficient than conventional ELISAs. Using the newly developed ECL method we showed, for the first time, that delta-hemolysin toxin can induce an immune response as antibody titers could be detected.

Enhanced anti-tumor immunity by superantigen-pulsed dendritic cells.

Staphylococcal enterotoxins A (SEA) and B (SEB) are classical models of superantigens (SAg), which induce potent T-cell-stimulating activity by forming complexes with MHC class II molecules on antigen-presenting cells. This large-scale activation of T-cells is accompanied by increased production of cytokines such as interferon-γ (IFN-γ). Additionally, as we previously reported, IFN-γ-producing CD8(+) T cells act as "helper cells," supporting the ability of dendritic cells to produce interleukin-12 (IL-12)p70. Here, we show that DC pulsed with SAg promote the enhancement of anti-tumor immunity. Murine bone marrow-derived dendritic cells (DC) were pulsed with OVA(257-264) (SIINFEKL), which is an H-2Kb target epitope of EG7 [ovalbumin (OVA)-expressing EL4] cell lines, in the presence of SEA and SEB and were subcutaneously injected into naïve C57BL/6 mice. SAg plus OVA(257-264)-pulsed DC vaccine strongly enhanced peptide-specific CD8(+) T cells exhibiting OVA(257-264)-specific cytotoxic activity and IFN-γ production, leading to the induction of protective immunity against EG7 tumors. Furthermore, cyclophosphamide (CY) added to SAg plus tumor-antigens (OVA(257-264), tumor lysate, or TRP-2) pulsed DC immunization markedly enhanced tumor-specific T-cell expansion and had a significant therapeutic effect against various tumors (EG7, 2LL, and B16). Superantigens are potential candidates for enhancing tumor immunity in DC vaccines.

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin‐18 secretion

Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus (S. aureus); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation. To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD. Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA. A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected (P < or = 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients (P < or = 0.05). A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.

Characteristics of Enterotoxin H–Producing Staphylococcus aureus Isolated from Clinical Cases and Properties of the Enterotoxin Productivity

Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.

Presence of IgE Antibodies to Bacterial Superantigens and Increased IL-13-Producing T Cells in Myelitic Patients with Atopic Diathesis

Background: Superantigens are considered to exacerbate autoimmune inflammation through the expansion of autoreactive T cells; however, the immune response to bacterial superantigens has not been extensively studied in any type of myelitis. We recently reported the occurrence of a distinct type of myelitis in patients with atopic diathesis, which in a recent nationwide survey was reported to be widespread in Japan. The aim of this study was to investigate the presence of IgE antibodies to bacterial superantigens and the proportion of IL-13- or IL-5-producing CD4+ or CD8+ T cells in patients with myelitis and atopic diathesis. Methods: Twenty-four myelitic patients with and 12 myelitic patients without hyperIgEemia, 28 patients with multiple sclerosis (MS) and 34 healthy controls were enrolled in this study. IgE antibodies to staphylococcal enterotoxins A (SEA) and B (SEB) in sera were measured using a liquid-phase enzyme immunoassay, and the intracellular production of IL-5 and IL-13 in peripheral blood CD4+ and CD8+ T cells was measured by flow cytometry. Results: The myelitic patients with hyperIgEemia showed significantly higher positive rates of serum SEA/SEB-specific IgE antibodies (41.7 and 62.5%, respectively) than the healthy controls (5.9 and 8.8%), patients with MS (0 and 21.4%) and those with normoIgEemic myelitis (0 and 0%). Moreover, IL-13-producing CD4+ T cells and CD8+ T cells increased significantly in the myelitic patients with hyperIgEemia compared to the controls, while IL-5-producing CD4+ or CD8+ T cells did not. Conclusions: The IgE response to staphylococcal superantigens is heightened in myelitic patients with atopic diathesis, which might contribute to increases in IL-13-producing T cells and thus the development of myelitis.

Frequency of superantigen-producing Staphylococcus intermedius isolates from canine pyoderma and proliferation-inducing potential of superantigens in dogs

This preliminary study investigated the potential role of staphylococcal superantigens in the pathogenesis of canine pyoderma. The staphylococcal enterotoxins A (SEA), SEB, SEC and SED, and the toxic shock syndrome toxin-1 (TSST-1) were assayed in isolates from skins of dogs with pyoderma. Culture supernatants from 25 of 96 isolates were positive for multiple superantigens, with SEA and SEC being the most frequently detected. In in vitro stimulation of canine peripheral blood mononuclear cells and quantitative flow cytometry revealed that low concentrations of SEA and SEB were potent stimulators of blastogenesis of T cells.

Focal encephalitis in the Lewis rat induced by intracerebral enterotoxin superantigen and amplified by activated intravenous splenocytes

The histological response after intracerebral (IC) injection of superantigen (SAg) was investigated in unprimed Lewis rats. The staphylococcal enterotoxins (SE) A (SEA), and E, but not B or saline, induced a variable perivascular inflammation in the injected hemisphere (6.7±6.1 cuffs per tissue section with SEA; mean±SD). Adoptive transfer of mitogen activated splenocytes (AS) augmented the response to SEA significantly (18.5±11.4; P<0.05). With or without AS transient bilateral perivascular cuffs were observed around the ventricles and in the corpus callosum up to 3 days after IC injection. The findings demonstrate that local expression of SAg in the brain can cause encephalitis, depending on the number of activated T cells in the circulation.

The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome.

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants' sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants < or = 2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4-6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.

The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants’ sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants ≤2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4–6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome.

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/ 19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (chi2 = 5.42, P < 0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in > 50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C 1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (χ 2=5.42, P<0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in >50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.

Microbiological analysis of ‘black pudding’, a Trinidadian delicacy and health risk to consumers

The prevalences of Salmonella, Escherichia coli and Staphylococcus aureus in black pudding which originated from local vendors and supermarkets in Trinidad were determined. The enterotoxigenicity of S. aureus strains and occurrence of 0157:H7 strains amongst E. coli isolates were also investigated. For the 100 black puddings each sampled from supermarkets and vendors, the mean total aerobic plate count (TAPC) per g was 1.8 × 10 7 ± 1.5 × 10 7 and 1.5 × 10 8 ± 2.3 × 10 8, respectively. E. coli was isolated from 56 (56.0%) black pudding samples from supermarkets with a mean count per g of 09.2 × 10 6 ± 7.9 × 10 6 compared to a prevalence of 79% (79 of 100) and mean count per g of 3.2 × 10 7 ± 4.7 × 10 7 for samples from local vendors. The difference between prevalences was statistically significant (P <- 0.001; χ 2). Only 1 (2.2%) of 45 strains of E. coli from supermarket-purchased pudding tested, was an 0157:H7 strain compared to 9 (13.6%) of 66 strains of E. coli from vendor-sold black pudding. The difference was not statistically significant (P ≥ 0.05; χ 2). Five (5.0%) of 100 black pudding samples from supermarkets yielded Salmonella, with S. ohio being the predominant serotype. For vendor-sold black pudding however, 11 (11.0%) samples were positive for Salmonella with a new serotype, S. unnamed (4,12:d —) being responsible for 50% (6 of 12) of isolates from this source. Forty samples each of black pudding from supermarkets and vendors were all (100.0%) positive for S. aureus with mean counts per g being 3.1 × 10 5 ± 8.8 × 10 5 and 3.3 × 10 6 ± 7.7 × 10 6, respectively. Overall, 27 (33.8%) of 80 strains of S. aureus tested were enterotoxigenic producing staphylococcal enterotoxins A(SEA), SEB, SEC, SED or a combination. It was concluded that black pudding poses a high risk to consumers based on the prevalence, microbial load and toxigenicity of the pathogens detected.

Characteristics of Staphylococcus aureus strains isolated from clinical and non-clinical human sources in Trinidad: Susceptibility to bacteriophages and antimicrobial agents, and toxigenicity

The susceptibility of Staphylococcus aureus strains isolated from human clinical and non-clinical sources in Trinidad to bacteriophages and antimicrobial agents was determined. The ability of the strains to produce enterotoxins and toxic shock syndrome toxin-1 (TSST-1) was also investigated. Of the 554 strains tested, 454 (81.8%) were susceptible to international phage set (IPS) phages with strains isolated from bacteruria (57.1%) and bacteremia (53.3%) having a low sensitivity compared to isolates from aspirates (87.3%) and anterior nares (97.4%). All sources combined, strains were most susceptible to phages belonging to several groups (mixed). Overall, 419 (75.6%) strains were resistant to one or more of nine antimicrobial agents tested. Resistance to penicillin was most prevalent, with 413 (74.5%) strains found to be resistant. Prevalence of resistance to tetracycline, gentamicin, oxacillin, cefuroxime and ciprofloxacin was 5.1%, 2.0%, 0.7%, 0.4% and 0.4%, respectively. Of the 554 strains tested, 307 (55.4%) produced staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) and D (SED) singly or in combination. Strains recovered from high vaginal swabs were least enterotoxigenic (40.0%) as compared to umbilical infection isolates which were most enterotoxigenic (78.9%). TSST-1 was produced by 95 (19.0%) out of 499 strains tested, with isolates from bacteruria found to be most toxigenic (33.3%). It was concluded that the S. aureus strains tested were highly susceptible to bacteriophages and antimicrobial agents (except penicillin) and that enterotoxigenic and TSST-1 producers were widespread and have an aetiologic potential.

Bacteriologic Quality of Some Trinidadian Ready-to-Consume Foods and Drinks and Possible Health Risks to Consumers

The bacteriologic quality of 10 ready-to-consume Trinidadian foods and drinks was determined by culturing for Staphylococcus aureus, Escherichia coli, Salmonella spp. and thermophilic Campylobacter spp. The toxigenicity of S. aureus and E. coli strains was determined and the susceptibility of S. aureus isolates to bacteriophages was also investigated. Of a total of 293 food samples tested, 58 (19.8%), 30 (10.2%), and 10 (3.4%) were positive for S. aureus, E. coli, and Salmonella spp., respectively. All samples were negative for Campylobacter but only two food types, chutney and preserved fruits, were negative in all pathogen assays. The sausage known as black pudding (boudin noir) was significantly (P ≤ 0.001; χ2 more contaminated with S. aureus (73.0%) than other foods and was also significantly (P ≤ 0.0l; χ2) more contaminated (43.2%) than all other foods except seamoss (33.3%) and roti (20.0%), where the difference was not significant (P ≥ 0.05; χ2). Black pudding also yielded all 10 strains of Salmonella isolated; the prevalent serotypes were S. agona (4 isolates) and S. derby (3 isolates). All 30 isolates of E. coli were negative for heat-labile enterotoxins (LT) and verotoxins (VT). Of the 58 S. aureus isolates, 33 (56.9%) were susceptible to bacteriophages with 19 (70.4%) of 20 black-pudding and 12 (66.7%) of 18 ice-cream isolates typeable. None of the isolates recovered from local sweets, snow cones, and roti was typeable. Regardless of the food source, strains of S. aureus were most susceptible to phages in various groups (mixed) constituting 21 (63.6%) of 33 strains typeable, followed by phages in groups III (18.2%) and V (15.2%). Twenty-two (37.9%) strains of S. aureus were enterotoxigenic with 8 (13.8%), 5 (8.6%), 13 (22.4%), and 12 (20.7%) strains producing, either singly or mixed, Staphylococcal enterotoxins A(SEA), B(SEB), C(SEC), and D(SED) respectively. It was concluded that black pudding, a local delicacy, poses the greatest health risk to consumers, making it imperative to institute sanitary measures during its production and sale.

Characteristics of Staphylococcus aureus strains isolated from bovine mastitic milk: bacteriophage and antimicrobial agent susceptibility, and enterotoxigenicity.

Staphylococcus aureus strains isolated from bovine mastitic milk in Trinidad were examined for their susceptibility to bacteriophages and antimicrobial agents and their ability to produce enterotoxins. Phage 42D was used to screen for bovine strains of S. aureus in milk. Of 250 strains tested, 224 (89.6%) were sensitive to phages in the international phage set (IPS), 85 (34.0%) were resistant to antimicrobial agents and 134 (53.6%) were enterotoxigenic. Strains lysed by phages in various groups (mixed) were prevalent, 145 (58.0%), followed by strains sensitive to groups III (17.0%) and I (8.8%) phages. A total of 72 (28.8%) strains were lysed by phage 42D either alone or with others. Resistance to penicillin was most common with 59 (23.6%) strains while 44 (17.6%) and 43 (17.2%) strains were resistant to ampicillin and triple sulphur respectively. Only 3 (1.2%) strains were resistant to methicillin. Prevalence of resistance to penicillin (12.5%) amongst phage 42D-sensitive strains was significantly (P < or = 0.01; X2) lower than for strains not lysed by phage 42D (28.1%) but strains susceptible to phage 42D were significantly (P < or = 0.05; X2) more resistant (4.2%) to methicillin than those not lysed by the phage (0.0%). Amongst 134 enterotoxigenic strains, 32 (23.9%), 77 (57.5%), 67 (50.0%) and 21 (15.7%) produced staphylococcal enterotoxins A(SEA), B(SEB), C(SEC) and D(SED) respectively either alone or mixed. SEB and SEC were significantly (P < or = 0.01; X2) more produced than either SEA or SED. Strains lysed by groups IV, i.e. 42D (62.5%), and III (56.7%) were more enterotoxigenic than those sensitive to phages in groups II (45.5%) and non-typable (46.2%) but the differences were not statistically significant (P > or = 0.05; X2). Strains lysed by group II phages (72.7%) were significantly (P < or = 0.05; X2) more resistant to antimicrobial agents than those lysed by phage 42D (18.8%). It was concluded that bovine mastitis strains of S. aureus in Trinidad were highly susceptible to bacteriophages and antimicrobial agents and enterotoxigenic and less than one-third may be considered to be bovine strains.

Bacteriological quality and associated public health risk of pre-processed bovine milk in Trinidad

The bacteriological quality of pre-processed raw milk originating from all 16 milk collection centres in Trinidad was evaluated. The mean total aerobic counts for bacteria, Staphylococcus aureus and Escherichia coli were determined. The pH and presence of somatic cells in milk were also determined. Of the 507 milk samples collected, 454 (89.5%) were California mastitis test (CMT)-positive and the mean pH was 6.50±0.13. The total aerobic plate count per ml was generally high for all samples ranging from 5.8 × 10 5 ±3.1 × 10 5 to 5.7 × 10 8 ± 1.5 × 10 9. S. aureus was isolated from 478 (94.3%) samples and the mean counts per ml ranged from 1.5 × 10 5 ± 1.1 × 10 5 to 9.4 × 10 5 ± 1.3 × 10 6. Nine (7.7%) of 117 strains of S. aureus produced staphylococcal enterotoxins A(SEA), B(SEB), D(SED) or a combination. E. coli was isolated from 105 (20.7%) samples and the mean counts per ml ranged from 6.6 × 10 2 ± 1.1 × 10 2 to 4.0 × 10 5 ± 4.6 × 10 5. Twenty-five (23.6%) of the 106 strains of E. coli tested produced verocytotoxin (VT) but none was positive for heat-labile (LT) toxin. It was concluded that pre-processed milk in Trinidad is of poor bacteriological quality. The detection of high counts of S. aureus in milk with some producing heat stable enterotoxins, coupled with the isolation of some verocytotoxigenic E. coli, are of public health significance to consumers.

Stimulation of tumor-draining lymph node cells with superantigenic staphylococcal toxins leads to the generation of tumor-specific effector T cells.

In animal studies, lymph nodes (LN) draining progressive tumors contain immunologically sensitized but functionally deficient T cells. These preeffector cells can differentiate into mature effector cells on stimulation in vitro with anti-CD3 and IL-2. However, anti-CD3 react with all T cells and the activated cell population expressed a broad but normal distribution of V beta phenotypes. In this study, we examined the feasibility of using bacterial superantigens to stimulate tumor-draining LN cells. Because of their TCR V beta restriction, superantigen activation may afford a means to identify T cell subsets that are important in the antitumor immune response. Stimulation of draining LN cells with staphylococcal enterotoxins A (SEA) or B (SEB) followed by culture in IL-2 resulted in selective activation and expansion of V beta 3 and V beta 11 or V beta 3 and V beta 8 T cells, respectively. However, in adoptive immunotherapy, SEB- but not SEA-activated cells mediated the regression of established pulmonary metastases. To define the relative antitumor effects of V beta 3 and V beta 8 T cells, SEB-activated cells were depleted of either V beta 3 or V beta 8 T cells with mAb and magnetic beads. The antitumor effects were demonstratably diminished after V beta 8 cell depletion but enhanced after V beta 3 cell depletion. Using antigenically distinct MCA 205 and 207 sarcomas, tumor regression mediated by the activated cells was found to be immunologically specific for the tumor that stimulated the draining LN. Furthermore, the SEB-activated cells were virtually all T cells consisting of approximately equal proportions of CD4+ and CD8+ cells and the collaboration of the two T cell subsets was required for in vivo antitumor effects. However, the helper function of CD4+ cells could be facilitated by the administration of exogenous IL-2. Despite their in vivo antitumor reactivity, SEB-activated cells did not exhibit tumor cytotoxicity in the 4-h 51Cr release assay. However, they secreted IFN-gamma on specific stimulation with tumor cells. Taken together, these results provide for the first time clear evidence of the functional significance of superantigen interactions with immunologically committed T cells and suggest a preferential V beta use that might be associated with the T cell immune response to progressively growing tumors.

In vivo antitumor efficacy of tumor-draining lymph node cells activated with nonspecific T-cell reagents.

Progressive growth of immunogenic murine tumors elicits a tumor-specific but functionally deficient T-cell immune response in the draining lymph nodes. These T cells, referred to as "pre-effector" cells could be induced in vitro to differentiate into mature immune effector cells, capable of mediating the regression of established metastases. Initially, tumor cells were used to stimulate the in vitro maturation of pre-effector cells. Alternatively, we found that pre-effector cells could be activated by sequential stimulation with anti-CD3 and interleukin-2 in the absence of tumor cells. In adoptive immunotherapy, these activated cells mediated therapeutic effects that were exquisitely specific to the tumor that triggered the pre-effector cell response in vivo. Since the anti-CD3 interaction with T cells is polyclonal, the activated lymph node cell population must also contain a significant number of T cells that do not have tumor specificity. In an attempt to selectively activate tumor-sensitized pre-effector cells, we recently utilized superantigenic bacterial toxins as T-cell stimuli for effector cell generation. Superantigens combine with major histocompatibility class II molecules to form the ligands that stimulate T cells bearing distinct T-cell receptor V beta elements. Lymph node cells draining the MCA 205 sarcoma stimulated with staphylococcal enterotoxins A (SEA), B (SEB), or C2 (SEC2) resulted in selective expansions of V beta 3 and 11, V beta 3 and 8, or V beta 8.2 T cells, respectively. Adoptive immunotherapy experiments revealed that SEB- and SEC2-, but not SEA- stimulated cells, mediated tumor-specific eradication of pulmonary metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal Condition for the Production of Unidentified Staphylococcal Enterotoxins

The sac culture method in combination with 6% NZ-Amine A plus 1% yeast extract was found to be the optimal condition for staphylococcal enterotoxins A (SEA) and D (SED) production. This growth condition was tested for the production of unidentified staphylococcal enterotoxins (SEs). Twenty-one Staphylococcus aureus strains that previously showed an emetic response in monkeys but were negative for any of the identified SEs when cultured by the membrane-over-agar method were grown by the sac culture method. All 21 strains produced at least one known SE. One strain produced only enterotoxin C (SEC). Nine strains produced only SED. One strain produced both SEA and SED. Four produced both SEC and SED. One produced SEA, enterotoxin B (SEB), and SED. Two produced SEA, SEC, and SED. Three produced SEA, SEB, SEC, and SED. One of these strains, FRI-569, that produced only SED at a low level (10 ng/ml) was selected to confirm the production of an unidentified SE by the monkey feeding test. Emesis was induced in five out of six monkeys. The total amount of SED in the supernatant fluids fed to monkeys was only 0.5 μg, or 2.5% of the 50% emetic dose (20 μg). Production of an unidentified SE by strain FRI-569 was therefore confirmed. This optimal condition can be used to produce increased amounts of unidentified SEs for purification.

Phage susceptibility, enterotoxigenicity and antibiograms of staphylococcus aureus strains isolated from human wounds and diarrhoea

The phage types, enterotoxigenicity and antibiograms of Staphylococcus aureus strains isolated from human diarrhoea and skin wounds in Nigeria were determined. Of 194 strains tested, 140 (72.2%) were typable using a combination of phages in the International Phage Set (IPS) for human strains of S. aureus and the bovine phage set. IPS phages lysed 118 (60.8%) strains while 96 (49.5%) were susceptible to bovine phages. S. aureus strains from adult diarrhoea were significantly (P less than or equal to 0.01; x2) more sensitive to bovine phages (52.8%) than to IPS human phages (16.7%). Strains isolated from wounds were however significantly (P less than or equal to 0.001; x2) more susceptible to IPS human phages (72.9%) than to bovine phages (41.5%). Phage group III strains were predominant amongst diarrhoeal isolates while wound strains were most susceptible to group II phages. Phage 119, a bovine phage, lysed all 19 strains of diarrhoeal origin sensitive to group M phages but all wound strains were resistant. Overall, 132 (68.0%) strains were enterotoxigenic producing staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) or a combination of these. A majority of diarrhoeal strains elaborated SEC while SEB production was predominant amongst wound strains. Of the eight antimicrobial agents used, S. aureus strains were most resistant to penicillin (71.6%) and least to sulfamethoxazole/trimethoprim (1.0%). Occurrence of resistance to one or more antibiotics was higher amongst wound strains (97.5%) than amongst strains isolated from diarrhoea (52.6%). It was concluded that bovine phage 119 may be a useful epidemiologic marker for S. aureus strains of bovine origin associated with human diarrhoea in Nigeria. It is however difficult to ascribe any aetiological significance to these strains because other enteropathogens not assayed for may have been present.