Production of Enterotoxin A

A method for production of enterotoxin A in multiple liter lots is described. The medium contained 4% N-Z Amine NAK supplemented with 0.001% niacin and 0.00005% thiamine, and was adjusted to pH 6. The inoculated medium in lots of 400 to 600 ml, in 2-liter Erlenmeyer flasks, was incubated at 37 C for 24 hr on a gyrotory shaker at 280 rev/min. Production of 4 to 6 μg of enterotoxin A per ml occurred.     

Modified method for production and purification of Staphylococcus aureus enterotoxin B.

A medium containing 4% bio-trypcase and 1% yeast extract was used for the production of Staphylococcus aureus enterotoxin B. The yield obtained was estimated at 200 micrograms of enterotoxin per ml of S. aureus S-6 culture supernatant. The purification method involves chromatography on Biorex 70 resin, isoelectric focusing, and gel filtration on Sephadex G-100. The purified enterotoxin (isoionic point, pH 8.55) was shown to be homogenous protein with a molecular weight of 29,000 when tested by gel electrophoresis.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.     

Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.     

Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.     

Production of Enterotoxin A in Milk

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 10(6)Staphylococcus aureus cells per ml. When similar milks were inoculated with 10(4)S. aureus cells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 10(6)S. aureus cells per ml and incubated at 35 C. Generally, a concentration of 5 x 10(7)S. aureus cells per ml of milk was reached before enterotoxin A was detected.     

Production of Staphylococcal Enterotoxins A, B, and C in Colloidal Dispersions

Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 mug of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken.     

Immunofluorescent Detection of Enterotoxin B in Food and a Culture Medium

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.     

Resistance of Free-Living Nematodes to Staphylococcal Enterotoxin

The usefulness of free-living nematodes for assaying staphylococcal enterotoxin was evaluated with a 98% pure enterotoxin B on five different nematodes. Included in the evaluation was an enterotoxin B in a crude culture filtrate. The filtrate of a culture of nonenterotoxigenic strains of Staphylococcus aureus, the uninoculated respective broth media, and distilled water were used as controls. The purified enterotoxin was found to exert no toxic effects at dosages ranging from 10 to 1,000 μg/ml for as long as 24 hr. Utilization of the toxin-protein by these nematodes was evidenced by their propagation after exposure times longer than 24 hr. The crude filtrate, containing 28 μg of enterotoxin per ml, was detrimental to nematodes to the same degree as the nontoxic filtrate and the uninoculated broths, in that they all caused irritation to external genitalia, motility changes, and death after comparable exposure times. This is in agreement with earlier observations that standard bacteriological fluid media, or broths containing over 1% protein hydrolysate or 1 to 2% salts, exert toxic effects on free-living nematodes.     

Effect of Sodium Chloride and pH on Enterotoxin C Production

Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 10(8) cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 10(8) cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 10(8) cells per ml and 6.38 to 7.30 for 3.6 x 10(6) cells per ml. In repeated experiments in which the inoculum contained 10(8) cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts.     

Staphylococcus aureus Enterotoxin B Release (Excretion) Under Controlled Conditions of Fermentation

Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation.     

Production of Staphylococcal Enterotoxins A, B, and C in Various Media

The effect of initial pH and of length of incubation time at 37 C in four different growth media on the production of staphylococcal enterotoxins A, B, and C was determined. A starting pH of 6.8 gave higher yields of enterotoxins B and C than either pH 6.0 or 5.3. The production of enterotoxin A was, however, not materially affected by the low initial pH of 5.3. Prolonged incubation (48 to 72 hr) resulted only occasionally in higher yields of enterotoxin. The effect of the media on the amount of enterotoxin produced is considerable. Difco Brain Heart Infusion (BHI) was inferior to either Fisher BHI, 4% NZ Amine (NAK), or 3% NAK plus 3% protein hydrolysate powder at the three initial pH values, regardless of length of incubation time. The slight effect of the low starting pH on the production of enterotoxin A is being further investigated.     

Repair and enterotoxin synthesis by Staphylococcus aureus after thermal shock.

To study repair and enterotoxin synthesis, four staphylococcal strains (FRI-100, FRI-137, FRI-472, and S6) were subjected to sublethal heat treatment, transferred to four liquid repair media (1% powdered skim milk in distilled water, complex medium, M9 minimal salt medium, and saline solution), and then incubated at different temperatures. Powdered skim milk proved to be the most efficient medium for promoting the repair of injured cells, particularly at 37 degrees C. Minimal salt medium also gave good results. Salt tolerance also increased at 4 degrees C, although it did not reach normal values. After 6 h of incubation at 37 degrees C in powdered skim milk, strain FRI-100 synthesized detectable amounts of enterotoxin A. After 10 h of incubation in the same medium at the same temperature, enterotoxins were detected in all of the strains.     

Amino Acid Requirements for the Production of Enterotoxin B by Staphylococcus aureus S-6 in a Chemically Defined Medium

A minimal chemically defined medium has been developed for growth (approximately 25 Klett units) and production of detectable enterotoxin B (approximately 5-6 mug/ml) by Staphylococcus aureus S-6. This medium contains monosodium glutamate as a source of carbon, nitrogen, and energy, three additional amino acids (arginine, cystine, and phenylalanine), six inorganic salts, and four vitamins. Increasing the concentrations of several amino acids in a series of defined media gave no increase in enterotoxin production. Apparently the limiting factor for growth and enterotoxin production in these media is the biosynthesis of one or more missing amino acids, rather than the concentration of the amino acids present in the media. An additional requirement for proline and valine was observed when glucose was added as the primary source of energy. When compared to complex media, our results indicated that the inhibitory effect of glucose on enterotoxin synthesis in defined media was less evident or totally absent.     

Scanning Isoelectric Focusing and Isotachophoresis of Clostridium perfringens Type A Enterotoxin

Fractionation of highly purified Cl. perfringens type A enterotoxin by scanning isoelectric focusing (SIF) and isotachophoresis (IT) in polyacrylamide gels is described for the first time. The use of 2% ampholytes pH 3–6 allowed the separation of enterotoxin into 2 species. The major component had an isoelectric point of 4·5 and possessed antigenic as well as functional activity. The minor component of enterotoxin, at equivalent concentrations, was devoid of any demonstrable biological activity had an isoelectric point of 4·6 and appeared to represent approximately 15% of the purified enterotoxin. With ampholytes pH 3·5–10 the minor and major components were focused at different times than when ampholine pH 3–6 was employed. Electrofocusing of enterotoxin in the presence of 6 M-urea did not alter the SIF pattern. During IT the major component of enterotoxin migrated ahead of the minor component. The 2 proteins were completely separated. Isotachophoretic separations required 0·023 M-phosphate pH 6·0 as the leading ion, 0·079 M-Tris as the counter-ion, 0·2 M-glycine (in Tris pH 8·1) as the terminating ion, 30 γ carrier ampholytes pH 3·5–10, 263 μg enterotoxin, 4% acrylamide and a current of 5 mA per gel column.     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

Sporogenicity of yeast autolyzates and casein hydrolyzates for Bacillus popilliae in liquid cultures

Bacillus popilliae NRRL B-2309S forms several million refractile spores/ml in liquid shaken cultures. A suitable medium includes glucose, K₂HPO₄, water, and three selected ingredients-activated carbon, a yeast autolyzate, and a casein hydrolyzate. Only 4 out of 26 lots of commercial yeast autolyzates tried were sporogenic. However, spore formation in the presence of the four was poor and erratic unless a compatible casein hydrolyzate also was present. Five of eight lots of casein hydrolyzates improved the sporogenicity of selected yeast products to various degrees. A comparison of amino acid compositions of yeast and casein lysates sheds no light on differences between suitable and unsuitable ones. Less refined yeast and casein products seem preferable.     

A procedure for extracting staphylococcal enterotoxin from foods at elevated temperatures and low centrifugal forces

Various low relative centrifugal forces (RCFs) were tested for their effectiveness in ‘cleaning up’ extracts of staphylococcal enterotoxin from food homogenates. RCFs of at least 10 000 × g were effective if centrifugations were for 30 min periods or longer. Below 8000 × g, and down to about 1000 × g, the cleaning up of extracts was effective only if centrifugation was preceded by filtration of food homogenates. Investigations of the thermal stability of enterotoxin during cleaning up of extract at higher than refrigeration temperatures showed that centrifugation temperatures of up to 40°C had little adverse effect on the serological activity of the toxin. Based on these findings a procedure was devised for extracting enterotoxin from foods using basic bench top centrifuge at ambient temperatures. Separation of enterotoxin from food proteins in the extract was achieved by carboxymethyl cellulose-column chromatography. Levels of enterotoxin A detectable by the microslide gel double diffusion method following extraction by this procedure were 2.5 ng/ml for milk, 2.5 ng/g for yogurt and 5 ng/g for cheese and corned beef.