Determination of staphylococcal enterotoxin A in cheddar cheese produced without starter activity.

Three variants of the chloramine-T radioiodination method were used to iodinate staphylococcal enterotoxin A with 125I. Only one method consistently produced usable labels for radioimmunoassay. The iodine incorporation was 55 to 76%; the specific activity was 3.5 to 5.5 muCi/microgram of enterotoxin, and the label was extremely stable on storage at -20 degrees C. Determinations of the enterotoxin in extracts of cheddar cheese produced without starter activity were carried out with the radioimmunoassay system and protein A as antibody immunoadsorbent. The assay buffer used in this system significantly influenced the detected levels of enterotoxin in the cheese extracts. Phosphate buffer, but not tris(hydroxymethyl)aminomethane (Tris) buffer, caused gelling of cheese extract proteins, thus resulting in an incomplete separation of free from antibody-bound 125I enterotoxin. When Tris buffer was used, the results indicated a high degree of accuracy and precision for this radioimmunoassay. The lowest detectable enterotoxin concentration in cheese extract was 0.5 ng/ml.     

Characterization of staphylococci isolated from mastitic cows in Spain.

A total of 57 gram-positive, catalase-positive cocci, considered etiological agents of clinical and subclinical bovine mastitis, were tested for glucose and mannitol fermentation, coagulase and thermonuclease production, sensitivity to lysostaphin, gelatin hydrolysis, lysozyme, phosphatase and egg yolk factor production, hemolytic properties, antibiotic sensitivity, susceptibility to human and bovine phages, and enterotoxin production. All 57 strains were identified as staphylococci. A good correlation was found between 3+ and 4+ coagulase reactions, thermonuclease production, and high sensitivity to lysostaphin. Neither mannitol fermentation nor production of other enzymes appeared to be a specific property of bovine Staphylococcus aureus strains. beta- and delta-hemolysins were more frequently found than alpha-hemolysin. Nearly 40% of the strains were penicillin resistant. Strains were lysed by phage 42E from the human phage set more frequently than by phage 42D, whereas with the bovine set, strains were more sensitive to specific bovine phages. Three strains produced enterotoxin C, and one strain produced enterotoxin D.     

Ecology of spontaneous fermentation in one winery during 5 consecutive years

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.     

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.     

Detection of antibodies to staphylococcal enterotoxins in the serum and milk of healthy goats

A study was made of the presence of antibodies (Ab) to staphylococcal enterotoxins A to E (SEA-SEE) in the serum and milk of 133 healthy goats, using a competitive ELISA method. Antibodies to some enterotoxins were detected in 83 sera (62.4%) and in 41 (30.8%) milk samples. In serum, antibodies to all SE types were detected, the most frequent being antibodies to SEA (24.8%). Milk contained antibodies to SEA, SEB and SEC, the latter being the most frequent (24.8%). A statistical study was performed to correlate the number of animals harbouring antibodies to a given enterotoxin with the presence in these animals of staphylococci producing that enterotoxin.     

Isolation and Partial Characterisation of Bacteria Recovered from Abscesses of Normally Slaughtered Pigs

One hundred and one pig abscesses of different localisation were examined. Bacteria were re-covered from all abscesses. In 6’8 abscesses a mixed aerobic and anaerobic flora was found. In 22 abscesses solely aerobes and in 11 solely anaerobes were found. Gorynebaoterium pyogenes and Fuso-bacterium necrophorum were found in 61 and 45 abscesses, respectively. Hemagglutination litres of G. pyogenes strains were higher with pig erythrocytes than with sheep erythrocytes. Three of 4 isolates of Staphylococcus aureus produced enterotoxin type A.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E.

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Studies on the functional site on staphylococcal enterotoxin A responsible for production of murine gamma interferon

To identify the functional region(s) associated with induction of gamma interferon on the staphylococcal enterotoxin A molecule, native staphylococcal enterotoxin A molecules and 12 various synthetic peptides corresponding to different regions of entire staphylococcal enterotoxin A were compared to induce gamma interferon production in murine spleen cells. The native staphylococcal enterotoxin A molecule induced gamma interferon production, whereas all of the 12 synthetic peptides did not. Pre-treatment of the murine spleen cells with synthetic peptide A-9 (corresponding to amino acid residues 161-180) significantly inhibited the staphylococcal enterotoxin A-induced gamma interferon production, whereas those with other synthetic peptides did not. When native staphylococcal enterotoxin A was pre-treated with either anti-staphylococcal enterotoxin A serum or anti-peptide sera, anti-staphylococcal enterotoxin A serum and antisera to peptides A-1 (1-20), A-7 (121-140), A-8 (141-160), A-9 (161-180) and A-10 (181-200) inhibited the staphylococcal enterotoxin A-induced gamma interferon production. From these findings, the amino acid residues 161-180 on the staphylococcal enterotoxin A molecule may be an essential region for murine gamma interferon production. Furthermore, the neutralizing epitopes may be also located on regions of amino acid residues 1-20, 121-140, 141-160 and 181-200 on the staphylococcal enterotoxin A molecule.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).     

SEROLOGICAL ATTRACTION OF NONTOXIC EGG COMPONENT TO STAPHYLOCOCCAL ANTI-ENTEROTOXIN

Raw whole liquid and dried eggs which were purported to contain staphylococcal enterotoxin were analyzed by a number of enzyme-linked immunosorbent assay (ELISA)-based methods. The initial evaluation was to establish whether the purported positive ELISA reactions were a result of toxin-anti-enterotoxin serological activity. A secondary consideration was to determine whether the putative protein occurred in the yolk and/or white portions of the eggs. A manual polyvalent detection system, manual monovalent ELISA and an automated polyvalent enzyme-linked fluorescent immunoassay were used to investigate this component in eggs. The ELISA results were instantaneous and showed strong positive reactions (false positives) with the manual and automated polyvalent systems, suggesting nonspecific binding of the egg-reactive component to one or more staphylococcal enterotoxin antibodies. Further analysis with the monovalent ELISA showed false-positive reactions when egg extracts were tested separately for enterotoxins A–E. The putative protein from fertilized eggs had caused false-positive reactions and the eggs did not contain preformed staphylococcal enterotoxin.

PRACTICAL APPLICATIONS

The analysis of raw whole liquid and dried eggs for staphylococcal enterotoxin must be performed with circumspection when applying serological systems which use whole antibody, as the resulting positive reaction could be a false-positive reaction due to a nontoxic component in eggs which reacts with the staphylococcal anti-enterotoxins.     

Characterization and cloning of enterotoxin genes of Salmonella typhimurium

Five of fifty five strains of Salmonella typhimurium of human origin was hybridized with both the LT-A and LT-B gene of Escherichia coli. The remarkably erythromatous and indurated response on rabbit skin and significant elongation of Chinese Hamster Ovary (CHO) cells indicated the production of enterotoxin of these isolates. The Salmonella enterotoxin is heat-labile and is not a secretory product. The LT gene of E. coli was used to analyze the chromosome and plasmid DNA from Salmonella typhimurium strains for toxin gene sequences. Southern blot analysis demonstrated that the toxin gene was located on the plasmid but not on the chromosome. Restriction enzymes BamHI, EcoRI, HindIII and PstI were used to analyze the DNA isolated from salmonella strains Nos.22, 52, 55 and 59. Three DNA fragments with size of 5.2 Kb of strain 22, 5.0 Kb of strain 52 and 8.6 Kb of strain 59 were identified as containing the enterotoxin gene. Plasmid pUC19 was used as the vector to clone these DNA fragments in E. coli. The rabbit skin permeability test indicated that Salmonella enterotoxin could be synthesized at readily detectable levels in these transformed E. coli.     

Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods.

Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A.     

Production and characterization of murine monoclonal antibodies against staphylococcal enterotoxins A and E

Six murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin A (SEA) and enterotoxin E (SEE) were prepared by fusion of myeloma cells with mouse spleen cells immunized with SEA and SEE. Of five MAbs to SEA tested, two MAbs were reactive with only SEA, whereas three were specific for both SEA and SEE. On the other hand, one MAb to SEE was found to be specific for only SEE. To study specificities of the combining sites of these MAbs, competitive binding assays with either SEA or SEE and horseradish peroxidase conjugated MAbs were performed using unconjugated MAbs as inhibitors. The results obtained in the assays suggest that different epitopes may be located on SEA and that some of them may be cross-reacting epitopes between SEA and SEE.     

Epidemiologic Typing of Methicillin-Resistant Staphylococcus aureus in Neonate Intensive Care Units Using Pulsed-Field Gel Electrophoresis

To elucidate the mode of dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in neonate intensive care units (NICUs), a total of 223 isolates from 3 separate hospitals were investigated between 1994 and 1996 by a DNA fingerprinting technique with pulsed-field gel electrophoresis (PFGE). Exoprotein profiles of some strains were also examined using SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) and the assessment of enzyme/toxin production such as coagulase, enterotoxin and TSST-1. Judging from the strain typing data from PFGE results and the epidemiological data, 2 different types of PFGE patterns (A and B) and their subtypes (A′, A″ and B′) were identified. The A type including A′ and A″ (comprising approximately 95% of the isolates) was markedly dominant. Only 5% of the isolates belonged to type B and subtype B′. On the other hand, MRSA isolated from adult patients admitted to the same hospital showed many different PFGE patterns. The results strongly suggested that some strain(s) with specific PFGE pattern(s) is prevalent in NICUs. Furthermore, isolates which expressed the same PFGE pattern did not always express the same SDS-PAGE pattern. There were some isolates with different abilities to produce coagulase, enterotoxin C and toxic-shock syndrome toxin (TSST)-1, and the abilities had no relation with a particular type of PFGE pattern. Therefore, a combination of PFGE analysis and biochemical analyses of coagulase, enterotoxin C and TSST-1 may provide us with more detailed information for the epidemiological study of MRSA in NICUs.     

Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy

Secondary and tertiary structural parameters of two functionally and serologically related proteins, staphylococcal enterotoxins B and C1, have been determined by using circular dichroism and fluorescence spectroscopy. The secondary structures derived from the respective far-UV circular dichroic spectra were 9.5% alpha-helix, 55.0% beta-pleated sheets, 16.5% beta-turns, and 19.0% random coils for enterotoxin B and 15.0% alpha-helix, 38.0% beta-pleated sheets, 25.5% beta-turns, and 21.5% random coils for staphylococcal enterotoxin C1. The values matched well with the secondary structures derived from the amino acid sequences (Chou and Fasman method). Seven antigenic sites have been predicted for both staphylococcal enterotoxins B and C1 by using the hydrophilicity and the secondary structure information. Three of these antigenic sites appear similar. Fluorescence quantum yield of the single tryptophan residue (Trp-197) of both the enterotoxins showed the tryptophan residue in staphylococcal enterotoxin B to be approximately 46% more fluorescent than in staphylococcal enterotoxin C1. Tryptophan fluorescence quenching by the surface quencher I- and the neutral quencher acrylamide revealed that the single tryptophan residue in each of the enterotoxins is buried in the protein matrix and is not accessible to the surface quencher I-. The tryptophan residue in staphylococcal enterotoxin C1 is 14% less accessible to acrylamide than in staphylococcal enterotoxin B. The data, in general, reflect several similarities and significant differences between the two related enterotoxins.     

Double antibody solid-phase radioimmunoassay for staphylococcal enterotoxin A

Summary A double antibody solid-phase (DASP) radioimmunoassay for staphylococcal enterotoxin A is described. In the assay the antigen-antibody complex is precipitated by anti-rabbit serum which is adsorbed onto a solid carrier (cellulose). The method is sensitive to 200 pg of enterotoxin. It was possible to detect as little as 2–5 ng enterotoxin A/ml food extract from minced meat and sausage. Enterotoxins B and C were not found to inhibit the uptake of labeled enterotoxin A at a level which might distort the results of the enterotoxin A assay. The DASP technique is sensitive, rapid, and easy to perform and thus compares favorably with other radioimmunoassays for enterotoxin.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Nature and reactivity of staphylococcal enterotoxin A monoclonal antibodies

Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.