[Occurrence and characteristics of staphylococcal enterotoxin antibodies in gynecologic patients]. / Výskyt a charakterizácia protilátok proti stafylokokovým enterotoxínom u gynekologických pacientiek.

OBJECTIVE: To control the occurrence of the antibodies against Staphylococcal type A and type B enterotoxin in gynaecological patients and in selected patients to determine the thermodynamic parameters of antibodies against Staphylococcal enterotoxins in their blood samples. DESIGN: Retrospective clinical study. SETTING: 2nd Department of Obstetrics and Gynaecology, P. J. Safárik University Kosice, Slovak Republic; Research Institute of Veterinary Medicine, Kosice, Slovak Republic; Department of Microbiology and Immunology, University of Veterinary Medicine, Kosice, Slovak Republic; Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, Madison, USA. METHODS: The occurrence of antibodies against Staphylococcal type A and type B enterotoxin was determined in 68 patients hospitalized in Department of Obstetrics and Gynecology in Kosice. RESULTS: The occurrence of antibodies against Staphylococcal enterotoxins was determined by radioimmunoassay (RIA) in blood samples of 45 (66%) patients. The antibodies against Staphylococcal type A enterotoxin were determined in 36 (53%) patients and the antibodies against Staphylococcal type B enterotoxin were determined in 9 (13%) patients. The antibodies against both type A and type B enterotoxins were determined simultaneously in blood samples of 10% of all patients. The thermodynamic parameters of the antibodies were determined in 5 patients with positive serum findings. CONCLUSION: With regard to the existence of heterogeneous clinical findings in large amount of patients with antibodies against Staphylococcal enterotoxins, the next study of Staphylococcal enterotoxins role in pathogenesis of wide spectrum of diseases is necessary.

Staphylococci from dental personnel.

Thirty dental students and five professors were cultured in nares, throat, and hands for the presence of staphylococci. Twenty-four students and two professors were colonized with staphylococci that were classified as S. aureus. Twelve students and one professor were colonized with staphylococci that produced enterotoxin. Care needs to be taken to avoid contaminating patients during dental examination, particularly during any type of surgery.

Controle de reaçöes inespecíficas na detecçäo de diminutas quantidades de enterotoxinas estafilocócicas em ensaio de aglutinaçäo passiva reversa em látex / Control of nonspecific reactions on reversed passive latex agglutination assay (RPLA) for detecting nanogram quantities of staphylococcal enterotoxins

Quatorze linhagens de estafilococos coagulase negativos procedentes de cabras sadias da Espanha, consideradas baixo-produtoras de enterotoxinas quando testadas por ELISA, e uma cepa de Staphylococcus aureus isolada do queijo envolvido em intoxicaçäo alimentar foram avaliados por RPLA, após crescimento em diferentes meios de cultivo. Reaçöes inespecíficas ocorreram para todas as linhagens e näo puderam ser eliminadas pela adiçäo de soro normal de coelho, novilha, carneiro e cavalo, nem por tratamento térmico das amostras-teste a 70ºC por 30 minutos. Entretanto, 5 por cento (v/v) de IgG normal e purificada de coelho adicionada aos sobrenadantes de culturas eliminaram as reaçöes inespecíficas

Prevention of nonspecific reactions on reversed passive latex agglutination assay (RPLA) for detecting low amounts of staphylococcal enterotoxins.

The SET-RPLA, from Denka Seiken Co. Ltd., Tokio, a commercial reversed passive latex agglutination test kit, has been recommended to establish the enterotoxicity capacity of some staphylococcal strains, implicated in food poisoning outbreaks that produce low levels of enterotoxins (SE). Despite the RPLA specificity, the occurrence of nonspecific reactions when testing low-SE-producing is common. In order to control these nonspecific reactions the addition of purified normal rabbit IgG purified was applied on approximately 350 staphylococcal isolates from human milk and anatomic sites of healthy dental student carriers. The results indicated that addition of 5% (v/v) of purified normal rabbit IgG (0.74 mg/mL) to the culture supernatant fluid is a simple and reliable tool for the controlling of nonspecific reactions in the RPLA assay.

Production of staphylococcal enterotoxin A in cream-filled cake.

Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10(6)) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210 degrees C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci.

Importance of staphylococci that produce nanogram quantities of enterotoxin.

Many staphylococcal strains produce enterotoxin, the toxin that is the cause of staphylococcal food poisoning. If a strain is enterotoxigenic it is possible for it to be involved in food poisoning. The gel diffusion methods were the first methods developed for detection of the enterotoxins and were thought adequate to detect their production. However, they were not adequate to detect enterotoxin in foods involved in food poisoning. When researchers began using the sensitive methods, such as enzyme-linked immunosorbent assay (ELISA) and reversed passive latex agglutination (RPLA), to check strains for enterotoxin production, some strains produced nanogram quantities of enterotoxin. When it was reported that several coagulase-negative species produced less than 10 ng/ml of enterotoxin, it was imperative to determine whether these strains produced enough enterotoxin in foods to cause food poisoning. At the present time research is under way to determine whether these strains produce enough enterotoxin in foods to cause food poisoning.

Staphylococcal food poisoning from cream-filled cake in a metropolitan area of south-eastern Brazil.

Twelve people became ill with vomiting and diarrhoea approximately four hours after eating cake with a cream filling at a birthday party and on the day following. The cake had been prepared by a food handler who had long experience in preparing foods for such functions. Staphylococcus aureus that produced enterotoxin A was isolated from the nose, the fingernails, and a healed infection on the neck of the food handler, and from the cake. Enterotoxin A was detected in the remaining portion of the cake. The cake, while still warm, had been refrigerated for one hour after it was prepared before it was removed for the party; it was refrigerated after the party. The cake was large (6 kg) and hence it was not adequately cooled in the hour during which it was refrigerated before the party. The conclusion is that the cake was accidentally contaminated by the food handler and inadequately cooled before it was eaten.

Production of staphylococcal enterotoxin D in foods by low-enterotoxin-producing staphylococci.

The goal of this investigation was to determine whether staphylococcal strains producing enterotoxins at nanogram levels per milliliter in laboratory medium, not detectable by gel diffusion methods, could produce sufficient enterotoxin in foods to result in food poisoning. Three low-enterotoxin D (SED)-producing strains were selected for this research because this enterotoxin is produced in smaller amounts than the other enterotoxins. The foods used were cream pie and cooked ham, divided into two portions, sterile and non-sterile. Each portion was inoculated with known concentrations of the staphylococcal strains under study and incubated for 48 h at 25, 30, and 37 degrees C. Samples were taken after 24 and 48 h. Enterotoxin was detectable in both sterilized and unsterilized cream and ham after 24 h at 37 degrees C with an inoculum of 10(3)/g. Some strains produced detectable amounts of enterotoxin in the sterilized foods after 24 h at 30 degrees C and some produced detectable amounts of enterotoxin in the sterilized foods after 24 h at 25 degrees C with inocula of 10(4)/g. It can be concluded that staphylococcal strains producing enterotoxin at ng/ml levels in laboratory medium, not detectable by gel diffusion methods, can produce sufficient enterotoxin (ng/g) in foods to cause food poisoning.

Symposium on microbiology update: old friends and new enemies. Staphylococcus aureus.

The analytical methods for the detection of the staphylococcal enterotoxins can be divided into 2 categories: (1) methods for detection of enterotoxin-producing staphylococcal strains; (2) methods for detection of enterotoxin in foods. Gel diffusion methods (Ouchterlony, microslide), in which the enterotoxin produced by any given strain is compared to one of the identified enterotoxins, are used most frequently for strain testing. The sensitivity of these methods is from 0.1 to 0.5 micrograms enterotoxin/mL, which is normally adequate to determine the enterotoxigenicity of strains. The methods for the detection of enterotoxin in foods need to be much more sensitive to detect less than 1 ng of enterotoxin/g of food that may be present. The radioimmunoassay (RIA), the enzyme-linked immunosorbent assay (ELISA), and the reversed passive latex agglutination (RPLA) method have the necessary sensitivity to detect 1 ng/g of enterotoxin in foods without the use of complicated extraction-concentration procedures. Kits based on the ELISA and RPLA methods are now available commercially for the detection of enterotoxins in foods. Tests have shown that the ELISA methods are somewhat more sensitive than the RPLA method.

Effect of environmental conditions on production of toxic shock syndrome toxin 1 by Staphylococcus aureus.

The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in a fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger.

Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle.

Synthesis of enterotoxins C1 and C2 and thermonuclease throughout the growth cycle was investigated with Staphylococcus aureus type strains FRI137 and FRI361 and S. aureus isolates M5 (C1) and L2 (C2) of animal origin. Both enterotoxins were produced during the exponential growth phase or at the beginning of the stationary phase. The minimal incubation time (7 to 12 h) and the lowest population (10(7) to 2 x 10(9) CFU/ml) associated with detectable enterotoxin (1 to 6.5 ng/ml) were related to the total amount of toxin produced after 24 h. Thermonuclease was detected in all samples whenever enterotoxins were detected. Furthermore, strain FRI137 produced thermonuclease earlier and at lower cell populations than it did enterotoxin C1. Patterns of enterotoxin and thermonuclease synthesis did not correlate. The concentration of toxins increased throughout the growth cycle, while the concentration of thermonuclease remained constant during the last hours of the growth cycle.

Production of a toxic shock syndrome toxin variant by Staphylococcus aureus strains associated with sheep, goats, and cows.

A toxic shock syndrome toxin (TSST) variant with an isoelectric point (pI) of 8.6 produced by an ovine-associated Staphylococcus aureus strain was described previously. Analysis of additional strains associated with sheep, goats, cows, and humans by isoelectric focusing with immunoblotting using monoclonal antibodies revealed that all 18 strains associated with sheep and all 12 strains associated with goats produced the TSST variant. Only 1 of 10 bovine-associated strains and no human-associated strains produced the variant, whereas the others produced TSST-1 (pI between 7.0 and 7.2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting indicated that both TSST-1 and the TSST variant had a molecular size of 24 kilodaltons.

Effect of chemical modification of histidine and tyrosine residues in toxic shock syndrome toxin 1 on the serologic and mitogenic activities of the toxin.

Modification of three or four of the five histidine residues in the toxic shock syndrome toxin 1 (TSST-1) with diethylpyrocarbonate did not inhibit the precipitin reaction of the modified TSST-1 with polyvalent antisera to the toxin. Monoclonal antibody 7T did not react with the modified TSST-1, but monoclonal antibody 8T did react with the toxin. Up to 50% of the mitogenic reaction of TSST-1 was inhibited by the histidine modification. Modification of one or two of the nine tyrosine residues in TSST-1 did not inhibit the precipitin reaction with polyclonal antisera to the toxin but did inhibit 85% of the mitogenic reaction.

Determination of biologically active region in toxic shock syndrome toxin 1.

The biologically active areas of toxic shock syndrome toxin 1 (TSST-1) were investigated using chemical fragmentation of the toxin with cyanogen bromide (CNBr). All three main CNBr-generated fragments of TSST-1, with estimated molecular weights of 20,000, 18,000, and 15,000, reacted with monoclonal antibodies (MAbs) 4T, 5T, 6T, 7T, 8T, and 9T, as determined by autoradiography. The epitopes involved in the mitogenic active site were located on the 15,000-Da internal fragment as determined by the neutralization of the mitogenic activity by the MAb. Modification of the methionine residues in the native TSST-1 by alkylation with iodoacetic acid had no effect on the serologic or mitogenic activity.