556 Modern Food Microbiology Figure 23–2 Enterotoxin B production, growth, and pH changes in Staphylococcus aureus at 37◦ C Source: From McLean et al.70 copyright c 1968 by American Society for Microbiology Figure 23–3 Rates of growth and enterotoxins A and B synthesis by Staphylococcus aureus S-6 Symbols:᭹, CFU/ml; , enterotoxin A; ᭡, enterotoxin B Source: From Czop and Bergdoll,27 copyright c 1974 by American Society for Microbiology Staphylococcal Gastroenteritis 557 exponential growth phase and at the beginning of the stationary phase.82 SEC1 was detected after 10 hours (2 ng/ml) at an S aureus population of 8.3 × 107 cfu/ml, while TNase was detected after hours with a cell count of 1.3 × 104 cfu/ml SEC2 and TNase first appeared after hours with a cell population of 107 cfu/ml.82 With both enterotoxins, TNase production ceased before enterotoxin production In regard to quantities of enterotoxins produced, levels of 375 and 60 µg/ml or more of SEB and SEC, respectively, have been recorded.91 In a protein hydrolysate medium, up to 500 µg/ml\l [Author: Please check the unit “µg/ml/l” here]of SEB may be produced.14 Employing a sac culture assay method, 289 ng/ml of SEA were produced by S haemolyticus, 213 ng/ml of SEC by S aureus, and 779 ng/ml of SED, also by S aureus.9 Chitin has been shown to enhance SEA production With 0.5% crude chitin in BIH broth, SEA production increased by around 52%.4 SEA thermostability was also increased, but cell growth was apparently not affected The production of SEH was favored by aeration and controlled pH with about 275 ng/ml having been produced in a fermentor at pH 7.0 with aeration at 300 ml/minute.102 Around µg/ml of SEG and SEI have been reported.76 SEB production in unbuffered media has been found to be repressed by excess glucose in the medium.74 Streptomycin, actinomycin D, acriflavine, Tween 80, and other compounds have been found to inhibit SEB synthesis in broth.38 SEB production is inhibited by 2-deoxyglucose, and the inhibition is not restored by glucose, indicating that this toxin, at least, is not under catabolite control.55 While actinomycin D has been shown to inhibit SEB synthesis in strain S-6, the inhibition occurred about hour after cellular synthesis ceased The latter was immediately and completely inhibited A possible conclusion from this finding is that the messenger RNA (mRNA) responsible for enterotoxin synthesis is more stable than that for cellular synthesis.62 The lowest number of cells of S aureus required to produce the minimum level of enterotoxin considered necessary to cause the gastroenteritis syndrome in humans (1 ng/g) appears to differ for substrates and for the particular enterotoxin Detectable SEA has been found with as few as ∼104 cfu/g.48 In milk, SEA and SED were detected with counts of 107 but not below this level.77 Employing a strain of S aureus that produces SEA, SEB, and SED, SEB and SED were detected when the count reached × 106 /ml and the enterotoxin level was ng/ml, while SEA at a level of ng/ml was detected with a count of × 107 cfu/ml.78 In imitation cheese with pH of 5.56–5.90 and aw of 0.94– 0.97, enterotoxins were first detected at the following counts: SEA at × 106 /g; SEC at × 108 ; SED at × 106 ; SEE at × 106 ; and SEC and SEE at × 106 /g.12 In precooked bacon, SEA was produced by strain A100 with cells >106 /g.95 In meat products and vanilla custard, SEA was produced with ≥log10 7.2 cells/g, but in certain vegetable products SEA production was delayed and detected only when numbers of cells were ≥log10 8.9/g.80 In the latter study, no SE could be detected in spinach and french beans after 72 hours at 22◦ C when cell numbers were log10 6.7–8.7/g All staphylococcal enterotoxins are resistant to pepsin (see exception in previous section) Mode of Action All staphylococcal enterotoxins, along with the toxic shock syndrome toxin (TSST), are bacterial superantigens (pyrogenic toxin superantigens—PTSags) relative to in vivo antigen recognition in contrast to conventional antigens With the latter, a CD4 T cell facilitates contact between T cell antigen receptors and major histocompatibility complex (MHC) class II molecules Staphylococcal superantigens bind directly to T cell receptor β chains without processing Once bound to MHC class II molecules, SEs stimulate helper T cells to produce cytokines such as the interleukins (IL), 558 Modern Food Microbiology gamma-interferon, and tumor necrosis factor Superantigens are thus proteins that activate many different T cell clones Among the cytokines, an overabundance of IL-2 is produced,60 and it appears to be responsible for many or most of the symptoms of staphylococcal gastroenteritis (see below) The activity of superantigens can be demonstrated in the laboratory by exposing murine splenocytes to SEs A positive response consists of T cell proliferation with concomitant production of IL-2 and gamma-interferon The administration of IL-2 produces many of the symptoms caused by the enterotoxin Studies with SEC1 concluded that SEC1 binds to an alpha helix of MHC class II such that the interaction between antigen-presenting cells and T cells is stabilized, leading to cytokine production and subsequent lymphocyte proliferation.51 The region of SEs responsible for emetic activity is unclear although this activity has been separated from superantigenicity (see reference 81) SEI-SEL appear to be only weakly emetic if at all Regarding the pathogenesis of enterotoxins in humans, many or most of the symptoms are caused by IL-2,60 including vomiting and diarrhea, and these symptoms can be produced by intravenous (IV) injections The C-terminus of the staphylococcal enterotoxin molecules is critical to several functions In one study using SEB, the deletion of only nine amino acids from this region led to complete loss of T cellstimulating activity.72 The C-terminus is believed to be critical to the three-dimensional conformation of the SEB molecule.72 Emetic and T cell proliferation activities can be disassociated When SEA was altered by deletion of three C-terminal residues, T-cell proliferation activity was retained, but the emetic activity was lost.52 Using mutant copies of SEA and SEB, it has been shown that the MHC class II binding property alone is not sufficient for emesis in monkeys.45 THE GASTROENTERITIS SYNDROME The symptoms of staphylococcal food poisoning usually develop within hours of the ingestion of contaminated food, although a range of 1–6 hours has been reported The symptoms—nausea, vomiting, abdominal cramps (which are usually quite severe), diarrhea, sweating, headache, prostration, and sometimes a fall in body temperature—generally lasting from 24 to 48 hours, and the mortality rate is very low or nil The usual treatment for healthy persons consists of bed rest and maintenance of fluid balance Upon cessation of symptoms, the victim possesses no demonstrable immunity to recurring attacks, although animals become resistant to enterotoxin after repeated oral doses.14 Because the symptoms are referable to the ingestion of preformed enterotoxin, it is conceivable that stool cultures might be negative for the organisms, although this is rare Proof of staphylococcal food poisoning is established by recovering enterotoxigenic staphylococci from leftover food and from the stool cultures of victims Attempts should be made to extract enterotoxin from suspect foods, especially when the number of recoverable viable cells is low The minimum quantity of enterotoxin needed to cause illness in humans is about 20 ng (see the outbreak reported in the section below) This value is derived from an outbreak of staphylococcal gastroenteritis traced to 2% chocolate milk From 12 cartons of milk, SEA was found at levels from 94 to 184 ng per carton, with a mean of 144 ng.34 The attack rate was associated with the quantity of milk consumed and somewhat with age; those aged 5–9 years were more sensitive than those aged 10–19 years Earlier findings indicated a dose of 20–35 µg of pure SEB for adults.88 From 16 incidents of staphylococcal gastroenteritis, SE levels of less than 0.01–0.25 µg/g of food were found.43 Staphylococcal Gastroenteritis 559 Table 23–7 Staphylococcal Foodborne Gastroenteritis Outbreaks and Cases in the United States, 1973–1987 Years 1973–1987 1983 1984 1985 1986 1987 Outbreaks Cases Percentage of All Cases 367 14 11 14 17,248 1,257 1,153 421 250 100 14.0 15.9 14.1 1.8 4.3 1.0 Source: Data from Bean and Griffin.10 INCIDENCE AND VEHICLE FOODS The incidence/prevalence of staphylococci in meats and seafoods are presented in Chapter (Table 4–3) and Chapter (Table 5–6) These organisms may be expected to occur in a wide variety of foods not given heat treatments for their destruction With regard to vehicle foods for staphylococcal enteritis, a large number has been incriminated in outbreaks, usually products made by hand and improperly refrigerated after being prepared Outbreaks and cases of foodborne gastroenteritis reported to the Centers for Disease Control for the years 1973– 1987 totaled 367 and 17,248, respectively (Table 23–7) From a high of around 16% in 1983, this syndrome accounted for only 1.0% of cases in 1987 The reported cases constitute only a small part of the actual number, however; estimates place the number of cases of staphylococcal foodborne gastroenteritis at between million and million per year in the United States The six leading vehicle foods for 1973–1987 are listed in Table 23–7, with pork and pork products accounting for more outbreaks than the other five combined One of the largest outbreaks ever recorded occurred in June–July 2000 in the Kansai District in Japan.7 There were 13,420 victims and the primary vehicle food was powdered skim milk from a single source An SE-producing strain of S aureus was the etiologic agent Symptoms appeared in 83.4% of interviewed victims within hours with 3–4 h being the peak period Vomiting was reported by 73.3% and diarrhea by 75.9% of victims A low-fat milk product contained ≤0.38 µg/ml of SEA, and powdered skim milk contained ca 3.7 ng/g.7 It was estimated that the average amount of SEA consumed/person was 20–100 ng For the years 1981–1995 in Korea, 64 outbreaks of staphylococcal food poisoning were recorded with 2,430 cases representing 16.5% of all foodborne outbreaks during this period.65 During the same period in Japan, 9.9% of all foodborne cases and 15.9% of outbreaks were staphylococcal.65 For the years 1980–1999 in Japan, there were 2,525 outbreaks of staphylococcal food poisoning and 59,964 cases with deaths (see reference 94) (see also Table 23–8) The leading vehicle foods were rice, rice balls, and bean curds; and SEA along with the combination of SEA and SEB were the most common enterotoxins Some other staphylococcal foodborne outbreaks are noted in Chapter 20 As noted in Chapter 22, the problem is one of reporting where all too often the small outbreaks that occur in homes are not reported to public health officials A large percentage of the reported cases of all types are those that result from banquets, generally involving large numbers of persons An unusual 560 Modern Food Microbiology Table 23–8 Leading Food Sources for Staphylococcal Gastroenteritis Outbreaks in the United States, 1973–1987 Food Sources Pork Bakery products Beef Turkey Chicken Eggs Number of Outbreaks 96 26 22 20 14 Source: From Bean and Griffin.10 outbreak was caused by SEA and SED and traced to wild mushrooms in vinegar.68 The food contained 10 ng SEA and ng SED per gram ECOLOGY OF S AUREUS GROWTH In general, the staphylococci not compete well with the normal biota of most foods, and this is especially true for those that contain large numbers of lactic acid bacteria where conditions permit the growth of the latter organisms (see Chapter 13) A large number of investigators have shown the inability of S aureus to compete in both fresh and frozen foods At temperatures that favor staphylococcal growth, the normal food saprophytic biota offers protection against staphylococcal growth through antagonism, competition for nutrients, and modification of the environment to conditions less favorable to S aureus Bacteria known to be antagonistic to S aureus growth include Acinetobacter,Aeromonas,Bacillus,Pseudomonas,S epidermidis, the Enterobacteriaceae, the Lactobacillaceae, enterococci, and others.75 SEA has been shown to be resistant to a variety of environmental stresses, but growth of several lactic acid bacteria did lead to its reduction and to a suggestion that toxin reduction might have resulted from specific enzymes or other metabolites of the lactic acid bacteria.24 PREVENTION OF STAPHYLOCOCCAL AND OTHER FOOD-POISONING SYNDROMES When susceptible foods are produced with low numbers of staphylococci, they will remain free of enterotoxins and other food-poisoning hazards if kept either at or below 40◦ F (4.4◦ C) or above 140◦ F (60◦ C) until consumed For the years 1961–1972, over 700 foodborne-disease outbreaks were investigated by Bryan19 relative to the factors that contributed to the outbreaks, and of the 16 factors identified, the most frequently involved were the following: inadequate refrigeration; preparing foods far in advance of planned service; infected persons’ practicing poor personal hygiene; inadequate cooking or heat processing; holding food in warming devices at bacterial growth temperatures ... SEA and SED were detected with counts of 107 but not below this level.77 Employing a strain of S aureus that produces SEA, SEB, and SED, SEB and SED were detected when the count reached × 106... ml/minute.102 Around µg/ml of SEG and SEI have been reported.76 SEB production in unbuffered media has been found to be repressed by excess glucose in the medium.74 Streptomycin, actinomycin D, acriflavine,... attack rate was associated with the quantity of milk consumed and somewhat with age; those aged 5–9 years were more sensitive than those aged 10–19 years Earlier findings indicated a dose of 20–35