A quantitative study of enterotoxin production by sheep milk staphylococci.

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Detection of staphylococcal enterotoxigenicity IV. Strains isolated in 1981 and 1982

Enterotoxin A, B, C, D and E detection and typing was undertaken in 807 staphylococcal strains isolated from food, breast milk, clinical material, diarrhoeal stools and hospital-collected swabs in 1981 and 1982. One hundred and sixty-six of the strains produced enterotoxin, most frequently type A or C, less so type D or B. There were single instances of strains with double toxin production: AB, AC or AD. Nine hundred and ten supernatants collected in 1972-1973 were additionally tested (after a lapse of 8 years) for type D enterotoxin; there were 152 positive specimens, predominantly relating to strains isolated from tinned cocoa and delicatessen, with 26 of the supernatants containing AD and BD enterotoxin combinations. For the first time the authors' laboratory detected strains producing enterotoxin F and the combination.     

Deoxyribonucleic Acid Base Composition and Biochemical Properties of Certain Coagulase-Negative Enterotoxigenic Cocci

Eight coagulase-negative, enterotoxigenic strains of cocci and one weakly coagulase-positive strain isolated from a number of different sources, including cases of food poisoning incidents, were evaluated for their relationship to Staphylococcus aureus on the basis of deoxyribonucleic acid (DNA) buoyant density and physiological studies. One strain of cocci produced enterotoxins A and C, two strains produced types B and C, four strains produced only type C, and one strain only type D. The enterotoxin produced by one strain of cocci was serologically untypable. None of the test organisms produced detectable amounts of enterotoxin in broth cultures. The test strains of cocci exhibited the following profile: all produced catalase; all grew anaerobically and fermented glucose; five were sensitive to lysostaphin; the percentage of guanine plus cytosine content of their DNA varied from 32.7 to 37.6; five produced acid from mannitol both aerobically and anaerobically; two formed δ-hemolysin; five produced phosphatase and acetoin; and all produced heat-stable nuclease. None of the organisms exhibited typical characteristics of S. aureus, S. epidermidis, or S. saprophyticus. On the basis of the present data and data reported elsewhere, these organisms should be considered as variants or mutants of S. aureus.     

Identification of a Fourth Staphylococcal Enterotoxin, Enterotoxin D

A fourth staphylococcal enterotoxin was identified serologically with antiserum to the very crude enterotoxic products of growth of a strain which also produces enterotoxin C, and then with antiserum to the considerably purified enterotoxic antigen of a strain which produces only the new enterotoxin. The identification of this antigen as enterotoxin D was based on the following observations. It was produced by strains which do not produce enterotoxins A, B, or C; it was absent in the growth products of nonenterotoxigenic strains; when appreciably purified, it was associated with emetic activity in the cat, and its biological activity was neutralized only by antisera containing its specific antibody and not by antibodies to enterotoxins A, B, and C. Staphylococcal strain 494 (ATCC 23235) was selected as the prototype strain. The production of this enterotoxin alone and together with enterotoxin A by strains of food-poisoning origin indicates that its role in food poisoning is second in frequency only to that of enterotoxin A. The incidence of production of enterotoxins A, B, C, and D, and of unidentified cat emetic substances by strains from several source categories, is presented.     

Identification of enterotoxigenic staphylococci from sheep and sheep cheese

The total of 127 Staphylococcus aureus strains obtained from sheep and sheep cheese were examined for their biochemical activities, biotypes, phage patterns, and ability to produce enterotoxins. Of the 83 staphylococcal strains isolated from animals 77 (93%) were classified as the C biotype. Of this group of sheep-adapted strains, 61 (79%) were sensitive to phage 78, and 46 (60%) produced enterotoxin C exclusively. The three isolated belonging to the A biotype produced enterotoxin D, and two of the three unclassifiable strains produced enterotoxin A. Of the 44 staphylococcal strains isolated from sheep cheese, there were 37 (84%) identified as the C biotype. From this series, 31 (84%) strains were lysed with phage 78, 6 (16%) strains produced enterotoxin C, and 1 strain produced enterotoxin A. One of the six strains determined as the A biotype produced enterotoxin D. C biotype strains, especially of ovine origin, are an exception among animal staphylococci, because a large number of them are enterotoixgenic. The C antigenic type is the most usual of the known enterotoxins in staphylococci of animal provenance.     

Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Factors Affecting the Secretion of Staphylococcal Enterotoxin A

The biosynthesis of enterotoxin A by replicating and nonreplicating cells was investigated. Unlike enterotoxin B, a secondary metabolite, enterotoxin A secretion resembled that of a primary metabolite by being secreted during the exponential phase of growth. The amount of toxin produced per unit of growth was not influenced by NaCl, NaNO(2), or NaNO(3). Several surfactants increased toxin secretion. Toxin secretion by nonreplicating cells was inhibited by chloramphenicol and 2, 4-dinitrophenol but not by streptomycin or penicillin G. The optimal pH for enterotoxin A production was 6.5 to 7.0. The findings suggest a number of possible reasons for the higher incidence of food poisonings caused by enterotoxin A as compared to enterotoxin B.     

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.     

Detection of low-enterotoxin-producing Staphylococcus aureus strains

Culture supernatant fluids from 26 (23.6%) monkey feeding test-positive Staphylococcus aureus strains, negative for enterotoxins by gel diffusion, were positive by enzyme-linked immunosorbent assay for one or more of the identified enterotoxins. Staphylococcal enterotoxin D (SED) was produced by 23 (88.5%) strains, SED and SEA were produced in two strains, and SED and SEC were produced in one strain. One strain produced only SEA, and two strains produced only SEC.     

Detection of low-enterotoxin-producing Staphylococcus aureus strains.

Culture supernatant fluids from 26 (23.6%) monkey feeding test-positive Staphylococcus aureus strains, negative for enterotoxins by gel diffusion, were positive by enzyme-linked immunosorbent assay for one or more of the identified enterotoxins. Staphylococcal enterotoxin D (SED) was produced by 23 (88.5%) strains, SED and SEA were produced in two strains, and SED and SEC were produced in one strain. One strain produced only SEA, and two strains produced only SEC.     

Enterotoxigenicity of Staphylococcus aureus strains isolated from poultry: raw poultry carcases as a potential food-poisoning hazard

Staphylococcus aureus strains were isolated from end-of-lay poultry carcases obtained from a plant at two different stages of processing before and after storage at different temperatures. These strains were supplemented with Staph. aureus strains isolated from poultry from a wide range of sources and biotyped, phage typed, and tested for production of enterotoxins A-E. The isolates were found to consist of poultry and human specific strains and each of these groups contained strains able to produce enterotoxin. Poultry strains produced only enterotoxin D whereas human strains produced enterotoxins A, C and D. The hen carcases used in storage experiments were found to be naturally contaminated with enterotoxin D producing staphylococci. No enterotoxin D could be detected on any of the carcases even after storage at temperatures which allowed multiplication of the organisms to occur (final Staph. aureus counts ranged from 10² to 10⁷/16 cm² of breast skin).     

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.     

Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Detection rate of enterotoxigenic Staphylococcus aureus isolated from children with intestinal disbacteriosis

Detection rate of enterotoxigenic Staphylococcus aureus isolated from faeces of 62 children aged from 3 months old to 7 years old with intestinal dysbacteriosis was studied by indirect hemagglutination assay and enzume immunoassay. It was shown that strains of S.aureus producing staphylococcal enterotoxin A (SEA) are prevailed (40.3%) in children with disturbances of intestinal microflora while staphylococcal enterotoxin B (SEB)-producing strains were detected in 20.9% of children. Amount of produced enterotoxin varied for SEA from 125 ng/ml to 2000 ng/ml and for SEB--from 125 ng/ml to 250 ng/ml. Inverse dependence of detection rate of enterotoxin-producing strains in faeces on age of children was established. The most number of enterotoxigenic strains of S.aureus was detected in infants. These data point to expediency of determination of enterotoxin-producing ability of S. aureus strains isolated from children with dysbacteriosis as measure of danger of this microorganism for children's health and indication for adequate actions for its elimination.     

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.     

Expression of Staphylococcus aureus enterotoxin type D in Escherichia coli X1776

The Staphylococcus aureus enterotoxin type D was cloned and expressed in Escherichia coli X1776 either as mature toxin or as a fusion with E. coli beta-galactosidase. Regulated expression was obtained and levels of toxin produced were in the order of 10(-3)-fold higher than in S. aureus.     

The Biochemical Activity of Enterotoxin and Non-Enterotoxin Producing Staphylococci

One hundred and sixty-nine staphylococcal strains of human origin have been tested for production of enterotoxin A, B or Ci, coagulase activity, DNase activity, typical growth on ETGP-agar, hemolysin production and the breakdown of mannitol under aerobic conditions. Very good correlation was observed between enterotoxin production and coagulase activity, in that 82 % of the enterotoxin producing strains also synthesized coagulase. The correlation between DNase activity and positive reaction in mannitol to enterotoxin production was also good (80 % of the enterotoxic strains produced both DNase and aerobic acid from mannitol). Of the enterotoxin producing strains 66 % hemolysed bovine erythrocytes and 61 % were ETGP-positive. However, the frequency of hemolysing respectively ETGP-positive but non-enterotoxin producing strains was very high, viz. 46 % respectively 32 %. It is concluded that enterotoxin production can not to a satisfactory degree of security be predicted by means of the other biochemical characters.     

Enterotoxigenicity of Salmonella strains of various origins

The study of the enterotoxigenicity of S. typhimurium with the use of the skin test on rabbits (to detect the delayed permeability factor) has revealed that these strains produce an enterotoxin similar to Escherichia coli thermolabile enterotoxin (TLE). Study of the enterotoxic activity of lysates obtained from 39 S. typhimurium strains and 5 S. dublin strains by sonication has revealed that 87% of S. typhimurium strains and all S. dublin strains produce an enterotoxin similar to E. coli TLE, as demonstrated by all tests used in this investigation, while 59% of S. typhimurium cultures and all S. dublin strains have been positive when tested for the capacity of producing the rapid permeability factor. "Hospital" strains and polyresistant cultures isolated from the environment (phagovar 20) are characterized by a higher rate of producing an enterotoxin similar to E. coli TLE, detected by the tests used in this investigation (90%), than antibiotic-sensitive strains of different origin (78%).