| CHAPTER 39 |

Staphylococcus aureus and Staphylococcal

Enterotoxins
Reginald W. Bennett, Jennifer M. Hait, and Sandra M. Tallent

39.1 INTRODUCTION owing to the presence of reservoirs in humans and healthy

animals.121 Staphylococcal species are ubiquitous and can
The proliferation of Staphylococcus aureus in food creates a
exist in the air, water, milk, or food, and on food contact
potential public health hazard, considering that many
surfaces or equipment.21 The primary reservoir for S. aureus
strains of S. aureus produce enterotoxins that may cause
in humans is the nasal cavity, and about 50% of adults are
food poisoning when ingested. Among the reasons for
carriers.74 Unless heat processes73 are applied, staphylococci
examining foods for S. aureus and/or staphylococcal
are expected to exist in low numbers in any or all foods that
enterotoxins are (1) to confirm that this organism may be
are handled directly by humans or are of animal origin.
the causative agent of foodborne illness; (2) to determine Some staphylococcal species, including both coagulase
whether a food or food ingredient is a potential source of negative and coagulase positive, are capable of producing
enterotoxigenic staphylococci; (3) to demonstrate post- highly heat-stable toxins that cause gastroenteritis in
processing contamination, which usually is due to human humans. S. aureus is the etiological agent predominantly
contact with processed food or exposure of the food to associated with staphylococcal food poisoning; however,
inadequately sanitized food-processing surfaces; and (4) to other staphylococcal species also have the ability to pro-
determine the presence or absence of pre-formed staphy- duce enterotoxins.13
lococcal enterotoxin in the product. Foods subjected to S. aureus is the cause of sporadic food poisoning episodes
post-processing contamination with enterotoxigenic types around the world. An epidemiological study should identify
of S. aureus represent a significant hazard because of the the suspect food and remove it from commerce, and then
absence of competitive organisms that normally restrict the determine the chain of events that permitted contamination
growth of S. aureus and the production of enterotoxins. by S. aureus in large enough numbers to cause illness.17
Staphylococcal food poisoning is responsible for an Accurate documentation of specific outbreaks is essential to
estimated 241, 148 illnesses, 1,064 hospitalizations, and 6 create epidemiological profiles. These profiles should
deaths in the United States each year.119 The true incidence include a history of the illness, including symptoms, detailed
of staphylococcal food poisoning is unknown for a number reports on those affected and information concerning the
of reasons, including poor responses from victims during suspect food.
interviews with health officials; misdiagnosis of the illness, Destruction of viable staphylococcal cells by heat does
which may be symptomatically similar to other types of not disable the activity of pre-formed staphylococcal
food poisoning (such as vomiting caused by Bacillus cereus enterotoxins. These toxins are highly heat stable and can
emetic toxin); inadequate collection of samples for labora- remain biologically active under a wide range of tempera-
tory analyses; and improper laboratory examination. ture conditions. Of the various metabolites produced by the
Despite underreporting, staphylococcal enterotoxins are staphylococci, the enterotoxins pose the greatest risk to
among the leading cause of foodborne illness. The most consumer health. Enterotoxins are proteins produced by
common symptoms of staphylococcal food poisoning are some strains of staphylococci,4,17,138 which, if allowed to grow
vomiting and diarrhea, which occurs 2–6 hr after ingestion in foods, may produce enough enterotoxin to cause illness
of the toxin. The illness is relatively mild, usually lasting when the contaminated food is consumed. These
only a few hours to 1 day; however, in some instances the structurally related, toxicologically similar proteins are
illness is severe enough to require hospitalization.14 produced primarily by Staphylococcus aureus. S. intermedius
There is a widespread distribution of staphylococci in and S. hyicus have also been shown to be enterotoxi-
the environment. Many of the 32 species and subspecies in genic.1,111,133,134 Normally considered a veterinary pathogen,
the genus Staphylococcus are potentially found in foods S. intermedius was isolated from butter blend and margarine

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in a food poisoning outbreak.13,78 A coagulase-negative S. for enterotoxin development is greater in foods that are
epidermidis was reported to have caused at least one exposed to temperatures that permit the growth of
outbreak.34 These incidents support testing staphylococci S. aureus. This is especially true for fermented meat and
other than S. aureus for enterotoxigenicity, if they are present dairy products. Although the potential is there, it is only
in large numbers in a food suspected of causing a food when improper fermentation takes place that the develop-
poisoning outbreak. ment of staphylococcal enterotoxin occurs.25
The need to identify enterotoxins in food encompasses In raw food, especially animal products, the presence of
two areas: foods that have been incriminated in foodborne S. aureus is common and may not be related to human
illness, and foods that are suspected of containing enter- contamination. Staphylococcal contamination of animal
otoxin. In the former situation, the presence of enterotoxin in hides, feathers and skins is common and may or may not
a suspect food confirms staphylococcal food poisoning. In result from lesions or bruised tissue. Contamination of
the latter, the presence or absence of the enterotoxin dressed animal carcasses by S. aureus is common and often
determines the marketability of the product. The latter unavoidable. Raw milk and unpasteurized dairy products
cannot be overemphasized because of the difficulty of may contain large numbers of S. aureus, usually as a result
preventing staphylococcal contamination of some types of of staphylococcal mastitis. Separating raw and processed
foods and food ingredients. Routine testing of certain types foods to prevent S. aureus cross-contamination is important
of foods for the presence of enterotoxins, however, is not the for food safety.
basis for good manufacturing practices. Because toxins are In foods in which S. aureus is destroyed by processing,
only discernible at levels of 106 Staphylococcus aureus cells/g the presence of S. aureus usually indicates contamination
of product, emphasis must be more rigidly placed on from the skin, mouth, or nose of food handlers. This
preventing the contamination and subsequent outgrowth of contamination may be introduced directly into foods by
S. aureus in food products. Symptoms of staphylococcal process-line workers with hand or arm lesions caused by
intoxication can occur after the ingestion of ,1 mg of toxin in S. aureus coming into contact with the food, or by coughing
a contaminated food product.26 In highly sensitive people a and sneezing, which are common during respiratory
dose of 100–200 ng can cause illness.55 The S. aureus popu- infections. Contamination of processed foods may also
lation at the time of analysis may be significantly different occur when deposits of contaminated food collect on or are
and not representative of the highest number of colony adjacent to processing surfaces to which food products are
forming units that occurred in the product. This should be exposed. When large numbers of S. aureus are encountered
taken into consideration when examining foods. in processed food, it may be inferred that sanitation,
Foods commonly associated with staphylococcal food temperature control, or both were inadequate.
poisoning are meat (beef, pork, and poultry) and meat The significance of the presence of S. aureus in foods
products (ham, salami, and hotdogs), salads (ham, chicken, should be interpreted with caution. The presence of large
and potato), cream-filled bakery products and dairy numbers of the organism in food is not sufficient cause to
products (cheese). Many of these items are contaminated incriminate a food as the vector of food poisoning. Not
after processing or cooking, when competing microorgan- all S. aureus strains produce enterotoxins. However, a
isms are eliminated. During preparation in homes or food- large population is indicative of the overall quality of the
service establishments they are subsequently mishandled food product. The potential for staphylococcal intoxica-
(e.g., improper refrigeration) prior to consumption. In tion cannot be ascertained without testing the enterotox-
processed foods, contamination may result from human, igenicity of the S. aureus isolate and/or, more
animal, or environmental sources. Therefore, the potential importantly, without demonstrating the presence of

Table 39-1. Summary of Staphylococcal Species Known to Produce Enterotoxins and Some Common Ancillary Tests74
Organism Enterotoxin Coagulase Hemolysis Nuclease Mannitol

S. aureus + + (+) TS (+)

S. intermedius + + + TS (+)
S. hyicus + (+) - TS -
S. caprae + - (+) TL -
S. chromogens + - - -w V
S. cohnii + - - - V
S. epidermidis + - V - -
S. haemolyticus + - + TL V
S. lentus + - - +
S. saprophyticus + - - - +
S. sciuri + - - +
S. warneri + - –w TL +
S. xylosus + - + - V

Note: + 5 positive; – 5 negative; –w 5 negative to weakly positive; (+) 5 weak reaction; TL 5 thermolabile; TS 5 thermostable;
V 5 variable.

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staphylococcal enterotoxin in food. Neither the absence isolation, or selective enrichment isolation, may be achieved
of S. aureus nor the presence of small numbers is by determining either an indicated number or the most
complete assurance that a food is safe. Conditions probable number (MPN) of S. aureus present. Common MPN
inimical to the survival of S. aureus may result in a procedures use three tubes or five tubes for each dilution.65,85
diminished population or the death of viable microbial The MPN procedure is recommended for surveillance of
cells, while sufficient toxin remains to elicit symptoms of products that are expected to have a small population of S.
staphylococcal food poisoning. aureus and a large population of competing organisms.
The method to be used for the detection and enumera- The direct plating method is suitable for analysis of
tion of S. aureus depends, to some extent, on the reason for foods in which a population of S. aureus is expected to be
conducting the test. Foods suspected to be vectors of .100 cells/g. For enumeration, samples may be applied to
staphylococcal food poisoning frequently contain a large a variety of selective media in two main ways: surface
population of S. aureus, in which case a highly sensitive spreading, and pour plates used in direct plating proce-
method will not be required. A more sensitive method may dures. Surface spreading is advantageous in that the form
be required to demonstrate an unsanitary process or post- and appearance of surface colonies are somewhat more
processing contamination, as small populations of S. aureus characteristic than the subsurface colonies encountered
may be expected. Usually, S. aureus may not be the with pour plates. The principal advantage of pour plates is
predominant species present in the food, and therefore that greater sample volumes can be used.17
selective inhibitory media are generally employed for Since the same types of selective media are frequently
isolation and enumeration. employed in both enrichment and direct plating, the
relative sensitivity of the two procedures depends largely
39.11 Staphylococcal Enterotoxins (SE) on the sample volumes. Larger volumes are normally used
Staphylococcal enterotoxins are single-chain proteins with in enrichment tubes, but equivalent volumes can be used in
molecular weights of 26-29 kDa.14 They are resistant to direct plating procedures by increasing the number of
proteolytic enzymes, such as trypsin and pepsin, which replicate plates. The plate count procedure is considered
allows them to transit intact through the digestive tract.14 more precise for the enumeration of S. aureus.14
There are seven classic enterotoxin serotypes: SEA, SEB, SEC1,
SEC2, SEC3, SED and SEE. Newly described SEs also 39.22 Media Commonly Used for Isolation
exhibiting emetic activity include SEG, she,130 and SEI.121,131
Selective media employ various toxic chemicals which
There are SE-like gene products that have not been confirmed
inhibit S. aureus to varying extents while also inhibiting
to exhibit emetic activity, and these are designated SElJ-
competitive species. The adverse effect of selective agents is
SElU.121 The different SE serotypes are similar in composition
and biological activity but different in antigenicity, and are more observable in processed foods containing injured cells
identified serologically as separate proteins.14 of S. aureus. A selective medium may help prevent the
The methods for identifying enterotoxins involve the overgrowth of S. aureus by competing species. The two
use of specific antibodies (polyclonal or monoclo- selective chemicals most frequently used in staphylococcal
nal).94,100,135,136 The fact that there are several antigenically isolation media are sodium chloride (NaCl) and potassium
different enterotoxins complicates their identification tellurite (K2TeO3). Various concentrations of these agents
because each one must be assayed separately. Another have been used, ranging from 5.5% to 10% NaCl and from
problem is that unidentified enterotoxins exist for which 0.0025% to 0.05% K2TeO3. Other chemicals, such as
antibodies are not available for in vitro serology. These ammonium sulfate, sorbic acid, glycine, lithium chloride,
unidentified toxins, however, appear to be responsible for and the antibiotic polymyxin, are frequently combined
only a small percentage of food poisoning outbreaks. with NaCl and K2TeO3. Sodium azide alone, or in
combination with NaCl and neomycin, has also been used
39.2 GENERAL CONSIDERATIONS FOR ISOLATION in selective isolation media. Researchers can further
OF ENTEROTOXIGENIC STAPHYLOCOCCI manipulate media by using combinations of selective
agents, changing pH, and including different combinations
S. aureus accounts for the preponderance of staphylococcal of diagnostic features.
gastroenteritis cases, although several other staphylococcal The principal diagnostic features of existing media
species have been recognized to produce enterotoxins.74 include (1) the ability of S. aureus to grow in the presence of
Described in Table 39-1 are some frequently used ancillary 7.5% or 10% NaCl37,42,85,88; (2) the ability to grow in the
tests to speciate enterotoxigenic staphylococcal species. presence of 0.01–0.05% lithium chloride and 0.12–1.26%
glycine,6,28,30,60,93,145,147 or 40 mg/mL polymyxin31,32,46,47; (3) the
39.21 Techniques for Isolation and Enumeration of ability of S. aureus to reduce potassium tellurite (K2TeO3),
S. aureus producing black colonies,4,6,30,125,147 both aerobically and
Enrichment isolation and direct plating are the most anaerobically93; (4) the form, appearance, and size of the
commonly used approaches for detecting and enumerating colony; (5) the pigmentation of colonies; (6) coagulase
S. aureus in foods. Enrichment procedures may be selec- activity and acid production in a solid medium29; (7) the
tive86,103,113 or nonselective.65 Nonselective enrichment is ability of S. aureus to hydrolyze egg yolk79; (8) the
useful for demonstrating the presence of injured cells,141 production of phosphates28,145; (9) the production of
whose growth is inhibited by the toxic components of thermonuclease8,33,48,83,108; and (10) growth at 42–43uC on
selective enrichment media. Enumeration by enrichment selective agar.91,141 Media used in the detection and

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enumeration of S. aureus may use one or more of these components of selective isolation media has not been
diagnostic features. demonstrated clearly. To distinguish between tube coagu-
lase-positive S. aureus and other tube coagulase-positive
39.23 Tests Used for Identification species, such as S. hyicus, tests for the presence of clumping
factor can be used. Clumping factor present in S. aureus cells
S. aureus is capable of producing a large number of
binds to the fibrinogen or fibrin present in human or rabbit
extracellular enzymes, toxins, and chemical components.
plasma, resulting in agglutination of cells. This is referred to
These extracellular metabolites have been useful in the
as slide coagulase, bound coagulation, or agglutination.
identification and isolation of S. aureus. Sometimes addi-
Clumping of cells in this test is very rapid (,2 min) and the
tional diagnostic features may be required to confirm
results are more clear-cut than 1+ or 2+ clotting observed in
S. aureus colonies, because the inhibitors used may not
the tube coagulase test. Clumping factor can be detected
completely prevent the growth of other organisms, such as
using commercially available latex agglutination
Gram-positive bacilli, micrococci, streptococci, and some
reagents.41,110 Anti-protein A immunoglobulin G (IgG) and
yeasts. Microscopic morphology helps to differentiate
fibrinogen are used to coat polystyrene latex beads to
bacilli, streptococci and yeasts from staphylococci, which
simultaneously bind protein A and coagulase, both of which
form irregular or grape-like clusters of cocci. Staphylococci
are specific cell surface components of S. aureus.
may be further differentiated from streptococci on the basis
Thermonuclease is a simple, rapid and practical test for
of the catalase test, with the former being positive.
routine identification of S. aureus.56,81,82,142 TNase is a heat-
Additional features are needed to further differentiate
stable phosphodiesterase that can cleave either DNA or
staphylococci from micrococci. Usually, staphylococci are
RNA to produce 39-phosphomononucleosides.21 Coagulase
lyzed by lysostaphin83,120 but not by lysozyme, and they can
and TNase tests are very efficient for the identification of
grow in the presence of 0.4 mg/mL of erythromycin.
foodborne S. aureus strains isolated on Baird–Parker
Micrococci are not lyzed by lysostaphin,83 may be lyzed by
agar.66,116 However, the use of the coagulase and/or the
lysozyme, and will not grow in the presence of erythromy-
thermonuclease test may result in erroneous species
cin. In a deep stab culture micrococci will grow at the
designation from a taxonomic standpoint. Two species, S.
surface, whereas most staphylococci grow throughout the
intermedius62 and S. hyicus51 subspecies hyicus, are both
agar. S. aureus will grow and produce acid from glucose and
coagulase and thermonuclease positive. However, the latter
mannitol anaerobically,7,132 whereas micrococci do not.
species can easily be differentiated from S. aureus on the
Staphylococcal cells contain teichoic acids in the cell wall
basis of the clumping factor test. Coagulase- and/or
and do not contain aliphatic hydrocarbons in the cell
thermonuclease- negative staphylococci are being reported
membrane, whereas the reverse is true with micrococci.5
to be enterotoxigenic.9,50,61,89
Further, the G + C content (mole percentage) of staphylo-
cocci is 30–40 and 66–75 for micrococci.5 Testing for some of
these features is difficult, time-consuming, and expensive, 39.3 SAMPLING REQUIREMENTS AND HANDLING
and is usually required only in special circumstances. OF SAMPLES
S. aureus is differentiated from the 32 other staphylo- Food and culture isolates to be analyzed should be kept
coccal species by a combination of the following features: refrigerated or frozen and should not be allowed to stand at
colony morphology and pigmentation; the production of ambient temperature except during processing. This is
coagulase, thermonuclease, acetone, b -galactosidase, phos- particularly true of foods that contain live organisms.
phates and a toxin (hemolysis); acid from mannitol,105
maltose, xylose, sucrose and trehalose; novobiocin resis- 39.31 Treatment of Samples
tance; presence of ribitol, teichoic acid, protein A and
The procedures for sample collection, shipment and
clumping factor in the cell wall.7,63,91 Several miniaturized
preparation described in the chapter ‘‘Sampling Plans,
commercial systems are available to speciate staphylo-
Sample Collection, Shipment, and Preparation for
cocci.84 The ultimate species identification may be estab-
Analysis’’ should be followed. Conclusions regarding the
lished by DNA–DNA hybridization with reference strains.
potential hazard of foods in commercial containers in
A nonisotopic DNA hybridization assay146 and a polymer-
which the presence of S. aureus has been detected should be
ase chain reaction procedure47,90 have been used to identify
drawn with considerable caution. Correlation of biotypes
S. aureus successfully.
isolated from food containers and from food poisoning
The confirmation procedures most frequently used to victims should be established.
establish the identity of S. aureus are the coagulase and
thermonuclease (TNase) tests.143 Coagulase is an enzyme
39.32 Handling Stock Cultures
that clots the plasma of human and other animal species.139
Differences in suitability among plasmas from various Stock cultures of the following properly identified organ-
animal species have been demonstrated.106 Human139 or isms should be maintained for testing the quality of media
rabbit plasma is most frequently used for coagulase testing and reagents:
and is available commercially. The use of pig plasma has
sometimes been found advantageous, but it is not widely N S. aureus (ATCC 12600) (a coagulase-positive biotype
available. Coagulase production by S. aureus may be affected with the combined characteristics of egg yolk hydrolysis
adversely by physical factors, such as culture storage and pigment production is preferable)
conditions or pH of the medium. The extent to which the N S. epidermidis (ATCC 14990)
production of coagulase may be impaired by the toxic N Kocuria varians (ATCC 15306)

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Storage of stock cultures on laboratory media that could media will prevent the growth of all competing species
result in desiccation of the media, thus requiring frequent without restricting the growth of some S. aureus. Among
transfer of stock cultures, should be avoided to lessen the the sources of variation shown to affect media efficiency
risk of losing certain diagnostic traits. significantly are (1) the type of food examined, (2) the
relative competitive position of S. aureus, and (3) the strain
39.4 PRECAUTIONS AND LIMITATIONS OF of S. aureus involved.46
METHODS The diagnostic criteria used in most staphylococcal
isolation media make visual colony identification of S.
Many factors affect the effectiveness and reliability of
aureus impossible without further testing. The physiology
S. aureus detection and enumeration procedures. Among
of S. aureus is diverse, and not all strains of the species
the more important are as follows (1) the physiological state
demonstrate similar activity because of their source. For
of the organism; (2) the competitive position of S. aureus in
example, not all biotypes have the capacity to hydrolyze
the sample; and (3) the limitations of isolation media.
egg yolk, a common diagnostic feature in many detection
The growth of injured S. aureus cells is restricted by and enumeration procedures.52,79,146 Considerable diver-
many of the selective isolation media used. Factors such as gence also has been demonstrated in the response of
heating, freezing, desiccation, ripening and storage, which various strains to the chemical agents used in selective
are common elements of food processing, have also been isolation media. This diversity may lead to considerable
shown to adversely affect the growth of S. aureus.7 The confusion regarding the suitability of various isolation
extent of cellular injury inflicted during processing media. Instability has been shown in some of the
depends on the type or severity of treatment. physiological traits demonstrated by this species.
Consequently, media satisfactory for detecting the presence Variability has been attributed to both physiological and
of S. aureus in animal lesions, excretory products and genetic factors. In applying the customary procedures for
nonprocessed foods may not be adequate for detecting detection and enumeration of S. aureus, possible variations
S. aureus in processed foods. in certain physiological traits should be considered.
The importance of the physiological state of S. aureus in
the selection of media for use in isolation and enumeration
39.41 Recommended Controls
procedures is receiving increased attention. Frequently
used staphylococcal isolation media that may restrict the Each batch of medium prepared for the isolation and
growth of sublethally heated cells are mannitol salt agar, enumeration of S. aureus should be tested for sterility,
egg yolk azide agar, phenolphthalein phosphate agar productivity, and the suitability of diagnostic criteria. To
containing polymyxin, milk salt agar, tellurite glycine test sterility, pour melted solid media into sterile plates and
medium,148 Staphylococcus medium number 110; and incubate 45–48 hr at 35–37uC. Liquid media also should be
tellurite polymyxin egg yolk agar.4,45,69,70,73,128,129 Selective incubated 45–48 hr at 35–37uC. Media productivity testing
media containing salt were more satisfactory than the may be accomplished by determining counts of S. aureus
media containing tellurite and tellurize azide in recovering obtained in 18–24-hr broth cultures grown in a noninhibi-
S. aureus presumably injured by the ripening process of tory medium such as brain–heart infusion (BHI) broth.
cheese.128 Metabolically impaired cells that survive the toxic Enumeration should be accomplished on a noninhibitory
chemicals of selective media also may fail to show typical solid plating medium such as BHI agar. The isolation
morphology. medium being tested for productivity should give counts
Agents used in media to improve the recovery of not significantly less (20%) than the noninhibitory medium.
stressed cells include61 (1) sodium pyruvate or catalase, Each prepared batch of medium should be streaked with
which acts to prevent cell death due to hydrogen peroxide known cultures of S. aureus to test for appropriate
accumulation during aerobic growth and repair35; (2) diagnostic characteristics, such as colony size and appear-
Polysorbate 80 for repair of damaged cell membranes ance, pigmentation, and egg yolk reaction. Each lot of
where lipid and phospholipid are located67; (3) a combined coagulase plasma or latex reagents should be tested with
supplement of 0.05% (w/v) Polysorbate 80 and 0.1% known cultures of S. aureus and S. epidermidis to determine
magnesium chloride hexahydrate (MgCl2N6H2O), where the suitability of the plasma for distinguishing positive and
Mg2+ may be required for repair of damaged ribosomes as negative reactions.
a consequence of Mg2+ loss after stress56,68,69,82; and (4)
phosphatidyl choline (2 mg/mL medium) or lecithin, 39.5 EQUIPMENT, REAGENTS, AND MEDIA
which acts similarly to egg yolk to increase the enumera-
tion of heat-injured S. aureus.4 39.51 Equipment and Supplies
The limitations of detection and enumeration methods
are generally those associated with limitations of the
N Glass spreading rods: sterile, fire-polished, hockey or L-
shaped, approximately 3–4 mm diameter, 15–20 cm long,
isolation media in supporting the growth of S. aureus and
with an angled spreading surface 45–55 mm long.
suppressing the growth of competing species. In addition
to variations contributed by the competition for growth N Drying cabinet or incubator for drying surfaces of agar
plates and for checking thermonuclease-positive colonies.
media nutrients, procedural efficiency may be affected by
other factors, such as acid–base changes and the production
of growth-limiting products, antibiotics, bacteriocins, bac- 39.52 Reagents
teriophages, and the microflora of food products. It is gen- N Coagulase plasma containing ethylenediamine tetraace-
erally conceded that none of the staphylococcal isolation tic acid (EDTA) (plasma derived from blood for which

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EDTA was used as the anticoagulant, or to which is 39.53. If growth is visible only on the bottom or sides of
added 0.1% EDTA (w/v). tubes, vortex-mix tubes before streaking. Streak plates to
N Commercial latex reagents for slide agglutination tests. obtain isolated colonies. Incubate 48 ¡ 2 hr at 35–37uC.
N Gram stain reagents. From each plate showing growth, pick at least one
colony suspected to be S. aureus and subject to coagulase,
39.53 Media clumping factor, or equivalent testing assay for confirma-
N Baird–Parker agar tion of S. aureus. Report MPN of S. aureus/g from tables of
N Baird–Parker agar containing rabbit plasma fibrinogen MPN values (see the chapter ‘‘Cultural Methods for the
N Baird–Parker agar without egg yolk Enrichment and Isolation of Microorganisms’’).
N Brain–heart infusion agar
39.63 Surface Plating Procedure72,103
N Brain–heart infusion broth
N Pork plasma fibrinogen overlay agar This procedure is recommended for the detection of S.
N Rabbit plasma fibrinogen agar aureus in raw, unprocessed food. The sensitivity of this
N Toluidine blue DNA agar procedure may be increased by using larger volumes
N Trypticase soy or tryptic soy agar (.1 mL) distributed over three plates.
N Trypticase soy or tryptic soy broth Prepare food samples by the procedure given in the
N Trypticase soy or tryptic soy broth (double strength) chapter ‘‘Sampling Plans, Sample Collection, Shipment,
N Trypticase soy or tryptic soy broth containing 20% and Preparation for Analysis.’’ Plating of two or more serial
NaCl dilutions may be required to obtain plates with the desired
N Trypticase soy or tryptic soy broth containing 10% NaCl number of colonies per plate.
and 1% sodium pyruvate For each dilution to be plated, distribute 1 mL of
sample suspension aseptically onto three plates of Baird–
39.6 PROCEDURES Parker agar or comparable media (e.g., 0.4, 0.3 and
0.3 mL). Spread the inoculum over the surface of the agar
39.61 Repair-Selective Enrichment Procedure65 using sterile, bent spreading rods. Avoid the extreme
This procedure is recommended for testing processed edges of the plate. Keep the plates in an upright position
foods likely to contain a small population of injured cells: until the inoculum is absorbed by the medium (about 10
Prepare food samples using the procedure described in min on properly dried plates). If the inoculum is not
the chapter ‘‘Sampling Plans, Sample Collection, Shipment, readily absorbed, plates may be placed in an incubator in
and Preparation for Analysis.’’ Transfer 50 mL of a 1:10 an upright position for about 1 hr before inverting. Invert
dilution of the sample into 50 mL of double-strength plates and incubate 45–48 hr at 35–37uC. Refer to Section
trypticase soy broth. Incubate 3 hr at 35–37uC. Add 100 mL 39.65 for alternative plating media, ideal when a shorter
of single-strength trypticase soy broth containing 20% incubation step is desired.
NaCl. Incubate for 24 hr ¡ 2 hr at 35–37uC. Transfer 0.1 Select a plate containing 20–200 colonies, unless only
mL aliquots of culture to duplicate plates of a Baird–Parker plates at lower dilutions (.200 colonies) have colonies
agar or comparable medium, and spread inoculum to with the typical appearance of S. aureus. If several types
obtain isolated colonies. Incubate the plates for 46 ¡ 2 hr at of colonies are observed that appear to be S. aureus, count
35–37uC. the number of colonies of each type and record these
Select two or more colonies suspected to be S. aureus counts separately. When plates at the lowest dilution
from each plate and subject to coagulase test or clumping plated contain ,20 colonies, they may be used. If plates
factor test. Report results as S. aureus present or absent in containing .200 colonies have colonies with the typical
5 g of food, as indicated by results of coagulase or appearance of S. aureus and typical colonies do not
clumping factor testing. appear on plates at higher dilution, use these plates for
enumeration of S. aureus, but do not count atypical
39.62 MPN Selective Enrichment Procedure71,103 colonies.
This procedure is recommended for detecting small Select one or more colonies of each type counted and confirm
numbers of S. aureus in raw food ingredients and S. aureus. Add the number of colonies on triplicate plates
nonprocessed foods expected to contain a large population represented by colonies confirmed to be S. aureus by biochemical
of competing species. verification using coagulase, clumping factor test, or other
Prepare food samples by the procedure described in the confirmatory test, and multiply by the sample dilution factor.
chapter ‘‘Sampling Plans, Sample Collection, Shipment, Report this number as number of S. aureus/g of product tested.
and Preparation for Analysis.’’
Inoculate three tubes of trypticase soy broth containing 39.64 S. aureus Colonies on Baird–Parker Agar6,103
10% NaCl and 1% sodium pyruvate at each test dilution S. aureus colonies are typically circular, smooth, convex,
with 1 mL aliquots of decimal sample dilutions. Maximum moist, 2–3 mm in diameter on uncrowded plates, gray-black
dilution of sample tested must be high enough to yield a to jet-black, frequently with a light-colored (off-white)
negative endpoint. Incubate 48 ¡ 2 hr at 35–37uC. margin, surrounded by an opaque zone and frequently with
Subculture all tubes that show growth. an outer clear zone; colonies have a buttery to gummy
Using a 3 mm inoculating loop, transfer one loopful consistency when touched with an inoculating needle.
from each growth-positive tube to dried Baird–Parker agar Occasional nonlipolytic strains may be encountered which
plates or other recommended media as described in Section have the same appearance, except that the surrounding

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opaque and clear zones are absent. Colonies isolated from Test is negative if no agglutination is observed in test circle.
frozen or desiccated foods which have been stored for Test is undistinguishable if agglutination is observed in
extended periods are frequently less black than typical both control and test circles. Strains showing irregular latex
colonies, and may have a rough appearance and dry texture. reaction must be confirmed by additional tests, as in
Section 39.7. Further instructions are provided in the kit
39.65 Alternative Plating Media insert.
Petrifilm (3M, St. Paul, MN) Rapid S. aureus Count Plate
contains modified Baird–Parker nutrients and a thermo- 39.68 Direct Enumeration of Coagulase-Positive
stable nuclease-reactive disk containing DNA, toluidine S. aureus
blue-O and a tetrazolium indicator that facilitates colony This method is specifically for use with all types of cheese,
enumeration and confirmation of S. aureus in 26 hrs.150
milk and dairy desserts.72 For each dilution of product,
BBL CHROMagar Staph aureus (Becton-Dickinson,
aseptically transfer 1 mL into a sterile 90 mm Petri dish.
Franklin Lakes, NJ) is a chromogenic medium that uses
Pour 10 mL melted rabbit plasma fibrinogen agar into each
an enzymatic reaction which generates mauve-colored
dish. Immediately after pouring the medium, mix the
colonies, with the growth of S. aureus in 24 hr.10
medium and inoculum using the following series of
Compact Dry X-SA (Nissui Pharmaceutical Co., Ltd,
motions: rock the plate back and forth five times, and then
Tokyo, Japan) uses a chromogenic substrate that forms
swirl the liquid in the plates five times clockwise followed
light-blue lenticular wet grazed colonies for S. aureus, and
by five counter-clockwise movements. Then allow the agar
small white or light-blue matte flat colonies for coagulase-
mixture to set. Incubate plates inverted at 35–37uC for 48 hr.
negative staphylococcus (CNS); results are obtained in
On rabbit plasma fibrinogen agar, S. aureus forms gray to
24 hr. This method has been AOAC performance tested
black colonies surrounded by an opaque or cloudy zone
method (PTM) tested and approved.96
indicating coagulase activity. Use plates for enumeration
RAPID Staph Agar (Bio-Rad, Hercules, CA) is a
that have between 10 and 100 typical colonies. Average the
medium based on an optimized Baird–Parker formula
number of typical colonies per dilution and multiply by the
which allows for the detection and enumeration of S. aureus
sample dilution factor. Report as S. aureus per g or mL of
in 24 hr.29 This method has been AOAC performance tested
product tested.
method (PTM) tested and approved.

39.66 Coagulase Test103 39.69 Direct Enumeration of Coagulase and

Thermonuclease-Positive S. aureus
With a sterile needle, transfer colonies to tubes containing
0.2 mL BHI broth and to trypticase or tryptic soy agar (TSA) This procedure is recommended for raw or processed
Slants. Incubate culture suspensions and slants 18–24 hr at foods. The sensitivity of the procedure may be increased by
35–37uC. Keep slant cultures at ambient temperature for plating larger inoculum volumes (.1 mL) distributed over
ancillary or repeat tests in case the coagulase test results are three or more plates.
questionable. Prepare two or more serial dilutions of food. Spread
Add 0.5 mL coagulase plasma with EDTA to 0.2 mL of 1 mL of sample suspension of each dilution equally over
each broth culture tube and mix thoroughly. Incubate at three plates of Baird–Parker agar without egg yolk,64 or
35–37uC and examine periodically over a 6-hr interval for Baird–Parker agar containing rabbit plasma–fibrinogen
clot formation. A 3+ or 4+ clot formation is considered a tellurite33,76 to which 0.5 mL of 20% sodium pyruvate66
positive reaction for S. aureus.112,126 Small or poorly was added just prior to use and then dried by incubating at
organized clots (1+ and 2+) should be confirmed by 50uC for 1 hr. Keep the plates in an upright position until
performing the ancillary tests listed below. Recheck doubt- the inoculum is completely absorbed at 35–37uC.
ful coagulase test results on broth cultures that have been If Baird–Parker agar containing no egg yolk is used,
incubated at 35–37uC for .18 hr but ,48 hr. Ensure culture dispense 8 mL of tempered pork plasma–fibrinogen over-
purity before rechecking coagulase test results. Do not store lay agar64 onto each plate. While this overlay is poured, the
rehydrated plasma for longer than 5 days at 2–8uC. plates must be on a horizontal surface. After the overlay
agar has solidified, invert and incubate plates for 45–48 hr
39.67 Clumping Factor by Latex Agglutination41 at 35–37uC.
Transfer a loopful of growth from an 18–24-hr trypticase or Select plates containing 20–200 colonies and count all
tryptic soy agar slant, or one or more colonies from Baird– black colonies showing the opaque fibrin haloes (coagu-
Parker agar to a control circle and to a test circle on a kit lase-positive) surrounding the colonies.33,64,76 Incubate these
reaction card. Add one drop control latex to control circle. plates at 65uC for 2 hr and then overlay each plate with
Add one drop test latex reagent to test circle. Mix contents 10 mL of melted toluidine blue DNA agar and allow to
of control circle with inoculation loop or wooden stick and solidify. After solidification, incubate plates at 35–37uC for
then, using the same loop or stick, mix contents of test 4 hr.
circle. Gently rock reaction card back and forth by hand for Count all colonies showing pink haloes against blue
about 1 min. Look for agglutination and significant clearing background as thermonuclease positive. Add all colonies
of the milky background under ambient light. Test positive that showed both fibrin haloes (coagulase-positive) and
and negative controls simultaneously with test cultures. pink (thermonuclease-positive) haloes, multiply by the
Test is positive if agglutination is observed in the test circle sample dilution factor and report as S. aureus per g or mL
and suspension in control circle remains homogeneous. of product tested.

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39.7 ADDITIONAL TESTS poisoning outbreak investigation, testing should be per-

formed to assess enterotoxin contamination in the food
39.71 S. aureus Speciation product and to determine the enterotoxigenicity of the
If anomalies are encountered during testing, additional staphylococcal isolates recovered. Currently the most
testing may be required to establish speciation of S. aureus. generally used method for the identification of staphylo-
The following tests are usually adequate. coccal enterotoxins is an enzyme-linked immunosorbent
assay (ELISA). There are several commercially available
39.72 Microscopic Examination ELISA kits that use both monoclonal and polyclonal
A Gram stain of S. aureus cultures will produce Gram- antibodies.18,20,137 The intensity of the color reaction or
positive cocci, 0.8–1.0 mm in diameter, occurring either fluorescence is proportional to the amount of toxin present
singly, in pairs, or most typically in irregular clusters in the sample.
resembling bunches of grapes.
39.81 General Considerations for the Detection of
39.73 Catalase Reaction Staphylococcal Enterotoxins
Emulsify growth from a TSA slant in 1 drop 3% hydrogen The minimum amount of enterotoxin required to cause illness
peroxide on a glass slide. Immediate bubbling is a positive in humans is not known. However, information from food
catalase test. Cultures of S. aureus are catalase positive. poisoning outbreaks27,55 and human challenge studies49
indicates that individuals experiencing illness probably
39.74 Production of Thermonuclease consumed at least 100 ng of enterotoxin A, the serotype most
Boil a portion of culture grown in BHI broth for 15 min and frequently involved in foodborne staphylococcal illness.39
use for thermonuclease test. Cut 2 mm or larger wells in A number of studies have been carried out comparing
toluidine blue DNA agar plates and fill with boiled culture enterotoxin detection methods,123,130,144 both in general and
growth using a Pasteur pipette. Touching the bottom of the specifically in foods,114 which were completed after studies on
well will usually draw enough liquid to fill the well to the characterization of purified enterotoxins.43,122 The mini-
level; if not, retouch the liquid with the pipette until the mum level of measurable enterotoxin with the microslide gel
well fills. It may be necessary to refill the pipette before double-diffusion technique40,149 is 30–60 ng/100g of food;
retouching. A trial test will indicate how much liquid to fill chromatographic102 and concentration procedures22 must be
the pipette with prior to touching the bottom of the well. used before serological assay. The microslide gel double-
Incubate plates at 35–37uC for 4 hr or 50uC for 2 hr. Colonies diffusion method40 is approved by AOAC International.
showing pink haloes extending 1 mm beyond the well are Several ELISA methods54,57–59,77,80,97,98,101,118,124,127 have been
considered positive for thermonuclease and considered to proposed to identify enterotoxins in foods. The VIDAS
be S. aureus. Include positive and negative controls using S. SET2 is an enzyme-linked fluorescent assay (ELFA)144
aureus (ATCC 12600) and S. epidermidis (ATCC 14990), technique (Figure 39-1) and the method of choice for
respectively. Use unboiled culture growth utilized for polyvalent systems to determine the presence of staphylo-
coagulase test. coccal enterotoxins SEA-SEE in foods and cultured isolates.
The second-generation antibody optimizes capture and
39.75 Susceptibility to Lysostaphin detection by using monoclonal anti-staphylococcal enter-
Mix 0.1 mL of cell suspension with 0.1 mL of lysostaphin otoxin antibodies and the removal of the sticky Fc region of
(dissolved in a 0.02 M phosphate buffer containing 2% the antibody, which allows for increased specificity by
NaCl) to give a final concentration of 25 mg lysostaphin/ reducing nonspecific binding that may cause a false positive
mL. To another portion of 0.1 mL cell suspension, add 0.1 reaction.15,16,75 The Tecra and other commercially available
mL of phosphate buffer with NaCl (negative control). Also visual enzyme immunoassay kits exist in the polyvalent and
include S. aureus (ATCC 12600) as a positive control and monovalent configuration. Several of these methods are
Micrococcus varians (ATCC 15306) as a negative control in presented in this chapter.
the assay. Partial or complete clearing of cell turbidity in TRANSIA PLATE Staphylococcal Enterotoxins
test and positive cultures with no clearing in the negative (Diffchamb, S.A. Lyon, France) is a sandwich enzyme
control is considered positive. S. aureus is lyzed (clearing) immunoassay for the detection of staphylococcal enterotox-
by lysostaphin. ins A, B, C1, C2, C3, D, and E in food and in bacterial cultures.
Reversed passive latex agglutination (RPLA)109 tests are
39.76 Utilization of Glucose and Mannitol commonly used to detect individual staphylococcal enter-
otoxin serotypes in a wide variety of foods and culture
Follow the procedures recommended by the Subcommittee isolates to give semi-quantitative results.109,123 Many com-
on Taxonomy of Staphylococci and Micrococci. Include mercially available kits commonly use latex particles that
controls of S. aureus (ATCC 12600) and M. varians (ATCC are sensitized with purified antiserum taken from rabbits.
15306). S. aureus will utilize glucose and usually mannitol The latex particle will agglutinate in the presence of the
anaerobically; M. varians will not.133 corresponding enterotoxin.
An alternative immunoassay method has been devel-
39.8 S. AUREUS AS AN AGENT OF FOODBORNE
oped by a number of researchers using bead-based
ILLNESS technologies.2,59,107 The fluorogenic beads are coated with
Staphylococcal enterotoxins are highly heat-stable proteins monoclonal staphylococcal enterotoxin antibodies washed
produced by some strains of S. aureus. During a food several times, and enterotoxin-positive food samples or

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 | Staphylococcus aureus and Staphylococcal Enterotoxins

Figure 39-1. Illustration of the VIDAS ELFA Technique.

cell-free culture supernatants are incubated with the antibody- from staphylococcal isolates or from food products. These
coated beads. The beads are washed again and incubated multiplex assays identify classic enterotoxin genes (SEA-
with a detection antibody and exposed to a laser reader that SEE) as well as some of the newly identified enterotoxins
measures the intensity of the fluorescence. The measurement (SEG-SEI, SER-SES) and enterotoxin-like enterotoxins (SElJ-
allows for the identification of staphylococcal enterotoxins SElQ, SElU-SElV).
or the identification of enterotoxigenic staphylococci.
Radioimmunoassay methods have also been used for 39.83 Enterotoxin in Foods25
the detection of staphylococcal enterotoxins.44
The major problem in identifying enterotoxin in foods is
the small amount that may be present in foods incrimi-
39.82 Enterotoxigenicity of Staphylococcal Strains
nated in food poisoning outbreaks. Marketable foods
The evaluation of staphylococci for enterotoxin production should not contain any enterotoxin. Toxins can be
is advantageous for identifying enterotoxin in foods and identified if the counts are (or at some time were) $ 106
desirable for examining strains isolated from various Staphylococcus cells/g. Such high counts are not acceptable;
sources. The methods outlined here are designed to therefore, instead of routinely testing products for the
determine the minimum amount of enterotoxin produced presence of toxins, the rules of good manufacturing
by a strain that could cause food contamination. practice emphasize the avoidance of contamination and
To determine the presence of enterotoxin in culture outgrowth of S. aureus.
fluid, latex agglutination or ELISA methods can be used. An additional problem may occur with pasteurized and
These commercial kits generally recommend broth media. thermally processed foods if toxins are rendered serologi-
It should be remembered, however, that S. aureus in pure cally inactive during processing.20 Methods have been
culture may occasionally produce substances that react developed and evaluated to restore serological activity to
nonspecifically with the immunoglobulin used. heat-altered toxin in extracts of heat-processed foods.3,11,12,19–22
Confirmation of the presence of a biologically active However, some current toxin detection assays are sensitive
enterotoxin should be considered the gold standard for enough to detect unaltered toxin that may persist after heat
evidence of food contamination, given that the detection of without such treatment if relatively large amounts of toxin
a gene does not prove the expression of a biologically active are present. To identify small amounts of toxin, a very
enterotoxin.50 Nevertheless, molecular DNA-based meth- sensitive procedure, or a satisfactory means of concentrat-
ods such as the polymerase chain reaction (PCR) technique ing the food extract, must be available. At the same time,
are useful alternative indicators, as they are rapid, reliable interfering substances must be removed from the extract.22
and sensitive in detecting gene targets. A standardized
molecular method99 could be a useful indicator of the
presence of enterotoxigenic staphylococci. The DNA-based
39.84 Injury
methods28,32 and phage typing30,31,117 would require further High heat such as retort (canning) temperatures can change
evaluation using more conventional testing methods, such the configuration of the enterotoxin protein, causing it to
as culture and enterotoxin assays. become serological negative. However, methods have been
There are several peer-reviewed articles in the literature developed to restore the serological activity to heat-
on conventional92,104 and real-time PCR87,95 assays used for processed foods.3,11,23,36 However, current toxin detection
the direct detection of the genes encoding enterotoxins methods may be sensitive enough to detect unaltered

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(retaining negative form) toxin that may persist after However, many of the recently described enterotoxins do
heating without treatment.3 not have antibodies commercially available for in vitro
serology.
39.9 EXAMINING STAPHYLOCOCCAL ISOLATES For raw or fermented foods and culture fluids from
FOR ENTEROTOXIN PRODUCTION staphylococcal growth in laboratory media, check after
Determining the enterotoxigenicity of S. aureus isolated extraction or collection of the culture fluid to determine
from food, food ingredients, or the food-processing whether the test preparation contains peroxidase, which
environment can be a significant step in predicting the could interfere with the proper interpretation of results. To
toxin serotype (A–E) in foods incriminated in foodborne determine the presence of peroxidase, add 50 mL of sample
intoxications. A number of methods38,53,115 have been to 50 mL of ELISA kit substrate reagent in an untreated
developed for the laboratory production of the staphylo- microtiter plate (no antibody to staphylococcal enterotoxin)
coccal enterotoxins. and let stand 10 min. If color changes to blue (or bluish-
green), the sample contains intrinsic peroxidase, which
39.91 Brain–Heart Infusion (BHI) Broth, pH 5.5 must be inactivated. If sample remains colorless (or original
color), then analyze it for enterotoxin by ELISA.
Enterotoxin production is described as follows: In order to inactivate intrinsic peroxidase, prepare a
30% (w/v) solution of sodium azide and add 1 mL of this
1. Special equipment, supplies, and media solution (30% w/v sodium azide) to 4 mL of test sample
a. Test tubes (final sodium azide concentration 6% [w/v]). Mix sample
b. Centrifuge with azide solution, add 50 mL/mL sample additive, and let
c. Centrifuge tubes stand 1–2 min at room temperature. Retest sample for
d. Media presence of peroxidase (50 mL sodium azide-treated sample
2. Preparation of materials with 50 mL ELISA kit substrate reagent), as described
a. Test tubes (25 6 200 mm) are used to sterilize above. If reaction is colorless (or original color), proceed
medium in 25 mL lots. Tubes containing BHI broth with ELISA to identify enterotoxin in the peroxidase-
may be stored at refrigerated or ambient tempera- inactivated sample.
tures until needed. When examining processed foods with obvious can
b. The culture medium normally used is BHI broth defects which might result in the growth of organisms that
pH 5.5, although other media such as 3% N-Z produce peroxidase, test the extract for peroxidase produc-
amine A plus 1% yeast extract are satisfactory. tion and inactivate as described above before testing for
3. Production of enterotoxin staphylococcal enterotoxin.
a. Inoculum: Pick representative colonies (5–10 for
each culture), transfer each to BHI broth (or 39.102 Equipment, Materials, and Reagents
comparable medium), and grow 18–24 hr at 35–
37uC (pH of culture should be approximately N Absorbent cotton
$8.0). N Blender for preparation of food extracts
b. Enterotoxin recovery: Centrifuge 10 min at 1000– N pH paper (range 0–14)
3000 g. Test the supernatant fluid for enterotoxin N Centrifuge and centrifuge cups
using a validated SE detection method. N Disposable plastic syringes (25 mL)
4. Selection of Desired Assay Protocol N Polypropylene tubes (12 x 75 mm)
a. Test 200 mL sample extract for 3M Tecra kit (3M N Polyethylene glycol (PEG, 15,000–20,000 mol wt)
Health Care, St. Paul, MN). N Dialysis tubing (12-14 kDa mol wt exclusion)
b. Test 100 mL for TRANSIA Plate Staphylococcal N Balance
Enterotoxins kit (Diffchamb, S.A., Lyon, France). N Beakers (250 mL)
c. Test 500 mL for VIDAS Staph Enterotoxin II N Tris buffer (0.25 M; 30.28 g TRIS/L, pH 8.0)
(bioMérieux, Marcy-l’Etoile, France). N Sodium hydroxide solution (1.0 N NaOH)
N Hydrochloric acid
39.10 EXTRACTION OF ENTEROTOXINS FROM N Deionized or distilled water
FOODS FOR ELISA SYSTEMS20,24 N Sodium hypochlorite
Tecra (3M, St. Paul, MN)
39.103 Procedures
Some recently developed rapid methods for identifying
enterotoxins in foods described in this chapter, with their 39.1031 Milk and Milk Powder
own more simplified extraction procedures, are the Reconstitute milk powder (25 g) by mixing with 125 mL
reversed passive latex agglutination; microtiter plate- 0.25 M Tris, pH 8.0. Treat reconstituted milk powder in
ELISA; polystyrene ball-ELISA; polyvalent (A-E serotypes) same way as fluid milk. For milk samples (5.0 mL), ensure
visual ELISA; automated immunoanalyzer; and the immu- that pH is in range 7–8; then add 50 mL of the sample
noenzymatic test. additive to 1 mL of eluate (when using the 3M Tecra kit).
For clearer extract, adjust milk sample to pH 4.0 with
39.101 General Precautions and Limitations concentrated HCl. Centrifuge sample for at least 10 min at
Methods for identifying the presence of preformed enter- 1,000–3,0006g. Decant extract and pump about 5.0 mL
otoxins have used monoclonal and polyclonal antibodies. through syringe containing wetted absorbent cot ton into

418 |
 | Staphylococcus aureus and Staphylococcal Enterotoxins

polypropylene tube. Readjust pH to 7.0–8.0 (use pH paper), 39.12 VIDAS STAPH ENTEROTOXIN II144
add 50 mL additive (in kit), and mix thoroughly. (bioMérieux Inc., Durham, NC)
39.1032 Dehydrated Food Ingredients This polyvalent assay is recommended to determine the
Add 125 mL 0.25 M Tris, pH 8, to 25 g of food, and allow presence of staphylococcal enterotoxins SEA-SEE in foods
the mixture to soak for 30 min. Homogenize in blender for and cultured isolates.140 This assay is AOAC approved
about 3 min at high speed. Centrifuge sample for about (AOAC Official Method 2007.06 VIDAS SET2 for Detection
10 min at 1,000–3,0006g and collect extract. Remove of Staphylococcal Enterotoxins in Select Foods, Final
plunger from plastic syringe containing pre-wetted absor- Action, 2010).75
bent cotton and carefully pump solution through, collecting
eluate. Take 5 mL of eluate; adjust pH to 7.0–8.0; then add 39.121 Materials and Reagents Supplied in Kit
50 mL of the sample additive to 1 mL of eluate (when using
the 3M Tecra kit), and mix thoroughly.
N 30 SET2 reagent strips containing enclosed wash solu-
tion, conjugate, and substrate
39.1033 Cheeses N 30 SET2 solid phase receptacle (SPRs): interior of the SET
Add 50 mL water to 25 g of cheese and homogenize for SPR coated with anti-enterotoxin antibodies
about 3 min at high speed in blender. Adjust pH to 4 (pH N 1 bottle standard (3 mL): purified staphylococcal
paper) with concentrated HCl. Centrifuge sample for about enterotoxin B (5 ng/mL) with 0.1% (w/v) sodium azide
10 min at 1,000–3,0006g. Remove plunger of plastic and protein stabilizers
syringe containing pre-wetted cotton, and place 5.0 mL of N 1 bottle positive control (6 mL): purified staphylococcal
extract into syringe; insert plunger and carefully pump enterotoxin B (5 ng/mL) with 0.1% (w/v) sodium azide
solution through, collecting eluate. Take 5 mL of eluate, and protein stabilizers; control range on the vial label
and add NaOH to adjust pH to 7.0–8.0; then add 50 mL of N 1 bottle negative control (6 mL): TRIS buffered saline
the sample additive to 1 mL of eluate (when using the 3M (TBS); polysorbate with 0.1% (w/v) sodium azide
Tecra kit), and mix thoroughly. N 1 bottle concentrated extraction buffer (55 mL): 2.5 mol/1
TRIS; 1% (w/v) polysorbate with 1% (w/v) sodium azide
39.1034 Other Foods
Foods other than those described above should be prepared 39.122 Materials Required for Procedure
as follows: Add 50 mL 0.25 M Tris, pH 8, to 25 g of food (Not Provided in Kit)
and homogenize for about 3 min at high speed in blender.
Centrifuge sample for about 10 min in bench centrifuge at N Pipette that will dispense a minimum of 0.5 mL
1,000–3,0006g. Remove plunger from plastic syringe N Tips, plastic, to deliver 500 ml
containing pre-wetted absorbent cotton and place 5 mL of N VIDAS automated immunoanalyzer
extract into syringe; insert plunger and carefully pump
39.123 Quality Control
solution through, collecting eluate in polypropylene tube.
Take 5 mL of eluate; adjust pH, if necessary, to 7.0–8.0; then Staphylococcal enterotoxins A, B, C1, C2, C3, D, and E are
add 50 mL of the sample additive to 1 mL of eluate (when detected by the VIDAS SET 2 assay at the sensitivity of at
using the 3M Tecra kit), and mix thoroughly. least 1 ng/mL.
Positive and negative controls are provided to validate
39.1035 Chloroform Extraction kit performance. Test the positive and negative controls
Treat the food extract with CHCl3 to remove lipids and with each new lot or shipment to ensure that assay
other substances that can interfere with the concentration of performance has remained unimpaired throughout ship-
the extract to small volumes.21 ping and storage. Test the controls as specified by your
laboratory’s regulatory guidelines. Controls are provided
39.1036 Selection of Desired Assay Protocol in ready-to-use form and must be thoroughly mixed and
N Test 200 mL sample extract for 3M Tecra kit (3M Health pipetted directly into the sample well of a reagent strip.
Care, St. Paul, MN). The expected positive control value will be included in
N Test 100 mL for TRANSIA Plate Staphylococcal the range indicated on the vial label. If the results from
Enterotoxins kit (Diffchamb, S.A., Lyon, France). testing the controls do not fall within this range, do not
N Test 500 mL for VIDAS Staph Enterotoxin (bioMérieux, report sample results. If the standard is out of range, the
Marcy-l’Etoile/France). test value can be recalculated with another standard. See
the VIDAS Operator’s Manual for complete information.
39.11 ELISA-BASED ENTEROTOXIN TESTING
Most ELISA-based assays as well as other serological 39.124 Procedure for VIDAS SET 2
systems use the whole antibody that has not undergone Prepare controls and extract enterotoxins from suspect
modification for the detection of staphylococcal enterotox- foods. In addition to the food extraction procedures
ins. The use of whole antibody (Fab1 + Fc fragments) has on described above, a greater variety of food extraction
occasion produced false positive results.16 However, an procedures are described by the kit manufacturer.
antibody has been produced which exhibits only the Fab1 Prepare food extracts immediately before testing.
fragment.75 Final action as stated below. This SET-II A standard must be run in duplicate for every lot of kits.
automated method has been used to rule out eggs as the The result is stored in the computer and used automatically
culprit in a food safety investigation.15 for assay analysis. A standard may be run with each SET2

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Table 39-2. Thresholds and Interpretations Results with test values less than the low threshold
indicate a sample without detectable enterotoxin. Samples
Test Value Threshold Interpretation
with test values greater than (or equal to) the high
,0.13 Negative threshold are reported as positive. Invalid results are
.0.13 Positive
reported when the background reading is above a
predetermined cut-off (indicating low-level substrate con-
tamination). In this case, repeat the assay with the original
sample.
An invalid result is also seen if there is no standard
work list, or a stored standard result may be used. See the available for the lot number of the sample test strip. In this
VIDAS Operator’s Manual for complete instructions. case, run a standard in duplicate in SET2 strips with the
same lot number as the invalid sample test. The sample test
1. Remove the VIDAS Staph enterotoxin kit from the result can then be recalculated using the new stored
refrigerator and allow it to come to room temperature. standard. See the VIDAS Operator’s Manual for complete
2. In the space provided, label the SET2 reagent strips information.
with the appropriate sample identification numbers.
3. Enter the appropriate assay information to create a 39.13 VISUAL ELISA: POLYVALENT (TYPES A–E)
work list. Type ‘‘SET’’ to enter the assay code, and SCREENING FOR DETERMINING
enter the number of tests to be run. If a standard is ENTEROTOXIGENICITY AND IDENTIFYING
being tested, type ‘‘S’’ (‘‘S’’ then ‘‘1’’ on mini VIDAS) STAPHYLOCOCCAL ENTEROTOXINS IN
for the sample ID. FOODS
4. Using a pipette 0.5 mL of standard, control or sample
into the center of the sample well of the SET2 reagent For staphylococcal enterotoxin (SET) visual immunoassay,
strip. ELISA performed in a double-antibody ‘‘sandwich’’ con-
5. Load the SET2 reagent strips and the SET2 SPRs into figuration (Figure 39-2). Capture antibodies specific for
the positions that correspond to the VIDAS section SET types A-E absorbed to plastic microtiter wells.23
indicated by the work list. Check to make sure the This visual immunoassay provides a rapid (4 hr),
color labels with the three letter assay code on the sensitive ($1.0 ng/mL or g) and specific screening test
SPRs and the reagent strips match. for the simultaneous identification of staphylococcal
6. Dispose of all used SPRs and reagent strips in enterotoxins A–E. The ELISA is performed in a ‘‘sandwich’’
appropriate biohazard containers. configuration. These kits are commercially available from
Tecra, 3M, as SETVIA96 in the polyvalent configuration
and as SIDVIA72 in the monovalent configuration for
39.125 Interpretation of Results
specific serotype identification of SEA, SEB, SEC, SED, and
Two instrument readings for fluorescence in the reagent SEE.20 The Tecra AOAC Official Method 993.06 received
strip’s optical cuvette are taken for each specimen tested. Final Action in 2000.
The first is a background reading of the cuvette and
substrate before the SPR is introduced into the substrate. 39.131 Equipment and Supplies
The second reading is taken after the substrate has been
exposed to the enzyme conjugate remaining on the interior Materials and reagents supplied in kit are listed as follows:
of the SPR. The background reading is subtracted from the
final reading to give a relative fluorescence value (RFV) for N Anti-SET antibody-coated Removawells (48 or 96 wells)
the test result. A test value is generated for each sample by N Removawell holder for securing wells
forming a ratio from the RFV of the sample to that of a N Instruction booklet methods manual
standard. Test values from both test and control samples N Color comparator
are compared to a set of thresholds stored in the computer. N Protocol sheet
Table 39-2 shows the thresholds and the interpreted results. N Wash concentrate
A report is printed that records the type of test N Sample additive
performed, the sample identification, the date and time, N Positive control, negative control
the lot number and expiry date of the reagent kit being N Conjugate diluent: conjugate, lyophilized
used, and each sample’s RFV, test value and interpreted N Substrate diluent: substrate, lyophilized
result. N Stop solution

Figure 39-2. Typical double-antibody ‘‘sandwich’’ ELISA scheme.

420 |
 | Staphylococcus aureus and Staphylococcal Enterotoxins

Materials, reagents, and equipment supplied by user are as remove residual liquid by firmly striking holder face-
follows: down on paper towel several times.
2. Transfer 200 mL aliquots of controls and samples (food
N Pipettes, 50–200 mL; 5–20 mL extracts or culture fluids) into individual wells; record
N Tips: plastic position of each sample on sample record sheet.
N Incubator: 35–37uC Gently tap holder containing test wells to ensure
N Plastic film wrap or sealable plastic container homogeneous distribution and contact of test material
N Microplate shaker (optional) with walls of wells. Agitation of wells on microtiter
N Microplate reader (optional, but dual wavelength plate shaker for 30 s is optional. To prevent evapora-
recommended) tion, cover wells with plastic film or plate sealers and
N Plastic squeeze bottle (500 mL) incubate 2 hr at 35–37uC. Wash liberally with wash
solution from squeeze bottles as follows: press
39.132 Preparation of Materials and Reagents Removawells firmly into holder. Quickly invert
holder, emptying contents into trough containing 2%
39.1321 Syringe-Type Filter (for Foods) (v/v) sodium hypochlorite. Remove residual liquid by
Prepare disposable plastic syringe (0.25 mL) by inserting firmly striking holder face-down on paper towel
plug of 0.5 cm thick absorbent cotton. Pump about 5.0 mL several times. Completely fill each well with wash
distilled water through to ensure tight packing. Do this just solution. Repeat liberal washing 2–3 more times.
before filtering 5 mL of food extracts for treatment with Finally, empty wells.
additive provided in kit. 3. Add 200 mL reconstituted enzyme conjugate into each
well. Cover tray and incubate 1 hr at room tempera-
39.1322 Reconstitution of Wash Solution ture (20–25uC). Empty wells and wash them thor-
Dilute wash concentrate with distilled or deionized oughly 5 times, as above. Empty wells and remove
water in reagent bottle to 2 L. Use this wash solution for residual liquid as described above.
washing wells and for diluting positive control when 4. Add 200 mL reconstituted substrate to each well.
required. Leave at room temperature (20–25uC) for at least
30 min until positive control reaches absorbance
39.1323 Reconstitution of Conjugate .1.0 or color darker than panel No. 4 on Color
Add conjugate diluent to conjugate and rehydrate at room Comparator. Color development tends to concentrate
temperature by mixing gently. around edges of wells. For accurate results, tap sides
of plate gently to mix contents before reading. Add
39.1324 Reconstitution of Substrate
20 mL of stop solution to each well. Tap sides of
Add substrate diluent to substrate.
plate gently to mix contents. Assay is now complete.
Determine results visually or with microtiter tray
39.133 Recommended Controls reader.
39.1331 Positive Toxin Control
Prepare by making 1:100 dilution of positive control solution
in wash solution (50 mL to 5 mL wash solution, as per kit 39.136 Interpretation of ELISA Results
directions) in a polypropylene tube. Run positive control
whenever assay is performed to indicate that all reagents 39.1361 Visual Observation
are functional and that assay has been conducted Place tray holding wells on white background, then
correctly. compare individual test wells with Color Comparator
provided in kit. Positive toxin control (and positive food
39.1332 Negative Toxin Control control, if used) should give strong green color, indicating
Use negative control solution provided in kit. No dilution that all reagents are functional. If negative control is
of negative control solution is necessary. Use 200 mL of all significantly darker than ‘‘negative’’ panels on Color
controls. Comparator, washing step was probably inadequate and
assay must be repeated.
39.134 Extraction of Toxin From Foods
1. Sample is considered positive when the following
1. See Section 39.10.
criteria are met:
2. Detailed extraction procedure described in the kit
a. Negative control is within negative range on Color
insert.
Comparator, and
b. Sample has green (or blue) color greater than
39.135 Tecra Procedure
negative range on Color Comparator.
1. Secure desired number of anti-SET antibody-coated 2. Sample is considered negative for enterotoxin when the
Removawells in holder provided. Allow 1 well for following criteria are met:
each food sample, 1 well for negative control, and 1 a. Negative control is within negative range on Color
well for positive control. Fill each well with wash Comparator, and
solution and let stand 10 min at room temperature (20– b. Sample is colorless or has color within negative
25uC). Empty wells by quickly inverting holder; range on Color Comparator.

| 421
Compendium of Methods for the Microbiological Examination of Foods |

39.1362 Absorbance Measurement With 11. Bennett, R. W. 1992. The biomolecular temperament of
Microtiter Tray Reader staphylococcal enterotoxins in thermally processed foods.
J. Assoc. Off. Anal. Chem. 75: 6–12.
Read absorbance (A) of samples at 414 ¡ 10 nm, using
12. Bennett, R. W. 1994. Urea renaturation and identification of
microtiter tray reader. Prepare dual-wavelength reader staphylococcal enterotoxin. In: R. C. Spencer, E. P. Wrights,
blank against air, and set second ‘‘reference’’ wavelength at and S. W. B. Newsom (eds.), RAMI-93: Rapid Methods and
490 ¡ 10 nm. Typical wavelength settings could be A405–490 Automation in Microbiology and Immunology. Intercept
or A414–492 for peroxidase-based systems such as the ELISA Limited: Andover, England.
described. Prepare single-wavelength instrument blank on 13. Bennett, R. W. 1996. Atypical Toxigenic Staphylococcus and
well containing 200 mL of substrate (provided in the kit) or non-Staphylococcus aureus species on the Horizon? An
water. Absorbance of positive toxin control should be at update. J. Food Prot. 59: 1123–1126.
least 1.0, indicating that all reagents are functional. If 14. Bennett, R. W. 2001. Staphylococcus aureus, In: R. G. Labbe
and S. Garcia (eds.), Guide to Foodborne Pathogens. John
absorbance of negative control is .0.200, washing of wells
Wiley & Sons, Inc: New York, NY. 201–220.
was probably inadequate and assay must be repeated. 15. Bennett, R. W. 2008. An antibody modified automated
Refer to Troubleshooting Guide in kit. enzyme-linked immunosorbent assay-based method for
detection of staphylococcal enterotoxin. J. Rapid Methods
1. Sample is considered positive if absorbance is .0.200. Autom. Microbiol. 16: 320–329.
2. Sample is considered negative if absorbance is #0.200. 16. Bennett, R. W. 2008. Serological attraction of nontoxic egg
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Generally, culture fluids that contain toxin have
17. Bennett, R. W., and G. A. Lancette. 2001. Staphylococcus
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