199

Journal of Food Protection, Vol. 65, No. 1, 2002, Pages 199–204

Research Note

Staphylococcus aureus Growth and Enterotoxin A Production in

an Anaerobic Environment
NEGASH BELAY AND AVRAHAM RASOOLY*

Division of Microbiological Studies, U.S. Food and Drug Administration, 200 C Street S.W., Washington, D.C. 20204, USA

MS 01-208: Received 15 June 2001/Accepted 18 July 2001

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ABSTRACT
The effects of strict anaerobic conditions on the growth of Staphylococcus aureus and the production of staphylococcal
enterotoxin A (SEA) were studied. The growth of S. aureus, a facultative anaerobic bacterium, is slower anaerobically than
aerobically. When grown on brain heart infusion broth at 378C, the anaerobic generation time at mid-log phase was 80 min,
compared with 35 min for the aerobic control. In contrast to previous studies demonstrating that staphylococcal cell density
was 9- to 17-fold greater in aerobic than in anaerobic cultures, data for a staphylococcal strain implicated in food poisoning
showed that the cell density was only two to three times as great in aerobic cultures. Production of SEA was monitored by
Western immunoblotting and shown to be growth dependent. With slower anaerobic growth, relatively less toxin was produced
than under aerobic conditions, but in both cases SEA was detected after 120 min of incubation. The combined effects of
temperature and aeration on S. aureus were also studied. Growth and toxin production of aerobic and anaerobic cultures at
temperatures ranging from 14 to 378C were analyzed. Growth was still observed at low temperatures in both environments.
A linear model for S. aureus aerobic or anaerobic growth as a function of incubation temperature was developed from these
studies. The model was tested from 17 to 35.58C, and the results suggest that the model can accurately predict the S. aureus
growth rate in this temperature range. The data suggest that anaerobic conditions are not an effective barrier against S. aureus
growth.

Staphylococcus aureus is a major cause of foodborne
illness around the world. Staphylococcal food poisoning re-
sults from ingestion of foods containing extracellular en-
terotoxins produced by the organism (3). Ten serologically
distinct staphylococcal enterotoxins (A, B, C, D, E, G, H,
I, J, and K) are currently known, and about 25% of S.
aureus strains isolated from foods produce enterotoxins (3,
8, 12). Many studies have been performed to evaluate the
effect of such environmental factors as pH, temperature,
substrate, oxygen concentration, and moisture content on
staphylococcal growth and/or enterotoxin production (3, 11,
12).
S. aureus is a facultative anaerobe, and the effect of
atmospheric composition (including N2 concentration, var-
ious CO2/O2 ratios, and vacuum) on staphylococcal growth
and enterotoxin production has been studied, with some
studies suggesting that staphylococcal cell density is 9- to
17-fold greater when S. aureus is grown under aerobic con-
ditions (6, 7). Staphylococcal enterotoxin A (SEA) produc-
tion under anaerobic conditions speciŽ cally in laboratory
media has been studied (1, 11, 13). However, these studies
provided qualitative measurements of end point toxin levels
(after 72 h) and did not assess SEA production in conjunc-
tion with staphylococcal growth kinetics. Indeed, few work-
ers (4, 5) have studied staphylococcal growth kinetics under
anaerobic conditions and carried out predictive modeling
* Author for correspondence. Tel: 202-205-4192; Fax: 202-401-7740;
E-mail: axr@cfsan.fda.gov.
for these conditions. A comprehensive model has been de-
veloped that takes into account the combined effects of
temperature, pH, NaCl, sodium nitrite, and aeration on
staphylococcal growth (4). However, for most practical pur-
poses, temperature is the primary variable parameter, and a
simpler model with temperature as the only variable may
be more useful.
The objective of this study was to evaluate the effects
of strict anaerobic conditions and incubation temperature
on growth and SEA production by S. aureus. The study,
by providing quantitative information for simple predictive
modeling purposes, will add to our understanding of the
kinetics of staphylococcal growth and SEA production and
may suggest factors that could be used to control S. aureus
contamination of foods.
MATERIALS AND METHODS
S. aureus ATCC 13565 (American Type Culture Collection,
Manasas, Va.), an enterotoxigenic strain implicated in food poi-
soning, was used in this study.
S. aureus growth analysis. The growth medium was brain
heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.)
and culturing of cells in a strict anaerobic environment was carried
out with the gassing apparatus, tubes, and procedures previously
described by Belay et al. (2). Cultures were incubated by shaking
(200 rpm) at the desired temperatures. Experiments were pre-
formed with tubes containing 5 ml of broth and an N2 gas phase.
Growth was measured as optical density (A600) with a Spectronic
21D spectrophotometer (Milton Roy Co., Rochester, N.Y.). Serial
200 BELAY AND RASOOLY J. Food Prot., Vol. 65, No. 1

captoethanol, and 0.01% bromphenol blue. The proteins were then

separated by SDS–polyacrylamide gel electrophoresis (SDS-
PAGE).

Exoprotein analysis. Exoprotein production was determined

by Coomassie blue staining of culture medium proteins separated
by SDS-PAGE, and SEA production was analyzed by Western
blotting (10). Proteins were separated on 12.5% SDS-PAGE gels
using a Bio-RDA mini gel (0.75 mm) apparatus. Gels were run
at 150 V for 2 h and were then electroblotted to a nitrocellulose
membrane (Nitroplus; MSI, Westbord, Mass.) at 400 mA for 60
min. The membrane was blocked with Tris-Tween 20 (10 mM
Tris [pH 8], 0.5% Tween 20, and 0.5 M NaCl) for 20 min and
incubated with rabbit anti-SEA (Sigma Chemical Co., St. Louis,
Mo.) diluted 1:300 in Tris-Tween 20. After incubation, the mem-

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FIGURE 1. The relationship between S. aureus optical density
measurements and CFU. Cultures were grown under aerobic con-
ditions at 378C. The optical density at 600 nm was measured and
serial dilutions of the culture were plated on BHI media to de-
termine the CFU.
dilutions of the culture were plated on BHI media to determine
the colony-forming units (CFU). Experiments were initiated with
a 0.2-ml inoculum from a culture grown aerobically to an optical
density of ;0.2.
Sample preparation. The culture broth (200 ml) was added
to 200 ml of loading buffer consisting of 0.25 M Tris-HCl (pH
6.8), 4% sodium dodecyl sulfate (SDS), 4% glycerol, 1% b-mer-
brane was washed and then incubated in goat anti-rabbit alkaline
phosphatase conjugate (Sigma) diluted 1:1,000 in Tris-Tween 20.
After washing, BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate
and nitroblue tetrazolium), an alkaline phosphatase conjugate sub-
strate solution (Sigma), was used as the color reagent.
RESULTS AND DISCUSSION
The effect of anaerobiasis on S. aureus growth and
exoprotein and SEA production. To study the effect of
an anaerobic environment on the growth and exoprotein
and SEA production of S. aureus ATCC 13565, cultures
grown aerobically and anaerobically at 378C were com-
pared. Culture growth was measured as optical density
(A600), and serial dilutions of similar cultures were plated
on BHI media to determine the CFU. The relationship be-

FIGURE 2. The effect of anaerobiasis on S. aureus growth and exoprotein and enterotoxin A production. S. aureus cultures were grown
under aerobic conditions or anaerobic conditions at 378C. (A) Growth curves of aerobic and anaerobic cultures. Closed symbols
represent aerobic cultures, and open symbols represent anaerobic cultures. (B) Coomassie blue–stained SDS-PAGE gel of S. aureus
exoproteins at various time points of the growth curve under aerobic (lanes 1 through 4) and anaerobic (lanes 5 through 8) conditions.
Lanes 1 and 5—after 120 min; lanes 2 and 6—165 min; lanes 3 and 7—250 min; lanes 4 and 8—435 min. (C) SEA production curve
under aerobic and anaerobic conditions. The relative amount of SEA produced at various time points was determined by densitometric
analysis of the SEA signal on Western immunoblots with a Hewlett Packard 4C scanner (Hewlett Packard, Palo Alto, Calif.). Closed
symbols represent the aerobic culture, and open symbols represent the anaerobic culture. (D) Western immunoblots of SEA at various
time points in S. aureus cultures grown under aerobic (lanes 1 through 4) and anaerobic (lanes 5 through 8) conditions. The blots
were developed with alkaline phosphate conjugated secondary antibody and measured by densitometry. Lanes 1 and 5—120 min; lanes
2 and 6—165 min; lanes 3 and 7—250 min; lanes 4 and 8—435 min.
J. Food Prot., Vol. 65, No. 1 S. AUREUS GROWTH AND ENTEROTOXIN A PRODUCTION 201

tween cell number and optical density for such cultures is

shown in Figure 1. As shown in Figure 2A, under aerobic
conditions the culture grew for approximately 200 min and
then entered the stationary phase. Anaerobic conditions
slowed culture growth, increasing the average generation
time during the mid-exponential phase of growth from ;35
to 80 min. Growth under anaerobic conditions also resulted
in decreased production of SEA (Figs. 1D and 2C). The
units in Figure 2C are scanner optical-density units and not
amount units; the graph shows the trend of toxin production
under each condition, rather than the absolute amount of
SEA produced. SigniŽ cant SEA levels, measured by West-
ern blotting, were detected after 120 min incubation under

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both conditions (Fig. 2D, lanes 1 and 5). Higher-molecular-
weight bands in Figure 2D are cross-reacting proteins, as
shown in our previous work (10).
S. aureus is also known to produce many other exo-
proteins, in addition to the enterotoxins, such as toxic shock
syndrome toxin 1 (TSST-1), proteases, and hemolysins.
Many of these extracellular proteins are known to be vir-
ulence factors (6, 9). Analysis with Coomassie blue–stained
gel (Fig. 2B) was carried out to examine the overall pro-
duction of extracellular proteins. A marked decrease in ex-
oprotein production in anaerobic cultures was observed.
This decrease was even greater than that observed for SEA.
Synthesis of several staphylococcal exoproteins and viru-
lence factors such as alpha-toxin, proteases, TSST-1, and
protein-A is controlled by the accessory gene regulator
(agr) (9). However, SEA is not agr regulated (9, 14), and
this may account for the difference we observed in patterns
of production of SEA and other exoproteins.
Barber and Deibel (1) observed decreased S. aureus FIGURE 3. The effect of an environment shift on S. aureus
growth and SEA production in BHI medium under anaer- growth. S. aureus cultures were grown under aerobic or anaer-
obic conditions, but only end point (72 h) data were ob- obic conditions for 150 min and were then switched to the op-
tained, and SEA was monitored qualitatively. The data pre- posite conditions. (A) S. aureus cultures grown under aerobic con-
sented here demonstrate that under favorable growth con- ditions and then shifted to anaerobic conditions. Open symbols
represent aerobic conditions prior to 150 min of incubation and
ditions, regardless of the presence of oxygen, a detectable
anaerobic conditions thereafter. (B) S. aureus cultures grown un-
amount of the toxin is produced after 120 min. However,
der anaerobic conditions and then shifted to aerobic conditions.
less SEA is produced under anaerobic conditions, consistent Closed symbols represent anaerobic conditions prior to 150 min
with previous work (3, 11) showing that the production of of incubation and aerobic conditions thereafter.
SEA is closely related to the extent of growth and is not
directly affected by culture conditions.
S. aureus toxin production is not always expected to toxin can be produced in 2 h whether or not oxygen is
follow cell number, although it does in this case; for ex- present.
ample, S. aureus TSST-1 production has been shown to To compare the effects of aerobic and anaerobic con-
vary independently of cell number under certain conditions ditions on S. aureus growth, experiments were carried out
(16). In a continuous-culture system, TSST-1 increased in which cultures were shifted from aerobic to anaerobic
from negligible peak levels in the presence of oxygen con- conditions or vice versa. As shown in Figure 3, shifts in
centrations of 1% or less to 500 ng/ml in the presence of growth conditions resulted in immediate changes in growth
2% oxygen. Toxin production then declined at higher ox- curve patterns. The growth rate of the anaerobic culture
ygen levels (16); it was also suggested that oxygen con- accelerated when the culture was shifted to aerobic condi-
centration itself may regulate the production of some toxins tions, and the growth rate of the aerobic culture slowed
(15). when the culture was shifted to anaerobic conditions. This
The fact that SEA can be produced early during both result suggests that bacterial growth rapidly adapts to the
aerobic and anaerobic growth explains why SEA is one of new conditions. Similarly, toxin production also changed
the most common foodborne toxins and is responsible for according to the shift imposed (data not shown), once again
many of the reported food poisoning outbreaks. In highly conŽ rming the relationship between SEA production and
contaminated food under favorable growth conditions, the growth conditions shown in Figure 2.
202 BELAY AND RASOOLY
FIGURE 4. The effect of temperature on S.
aureus growth under aerobic or anaerobic
conditions. Closed and open symbols rep-
resent growth of aerobic and anaerobic
cultures, respectively. (A) Growth at 148C.
(B) Growth at 208C. (C) Growth at 268C.
(D) Growth at 318C. (E) Growth at 348C.
(F) Growth at 378C.
The effect of temperature on aerobic or anaerobic
S. aureus growth. S. aureus can grow in a temperature
range of 10 to 458C (11). To assess the combined effects
of anaerobic conditions and temperature on S. aureus cul-
tures, growth and toxin production at temperatures ranging
from 14 to 378C were analyzed. As shown in Figure 4,
while anaerobic growth was lower than aerobic growth at
all temperatures, lower temperatures reduced growth in
both environments. However, the difference in generation
time between aerobic and anaerobic cultures was consid-
erably greater at higher temperatures than at lower temper-
atures. This difference suggests that the temperature is the
main determinant of the S. aureus growth rate under either
aerobic or anaerobic conditions.
At all temperatures tested in this study, the cell density
of S. aureus ATCC 13565 was two to three times as great
in the aerobic environment as in the anaerobic environment.
These results are signiŽ cantly different from those of pre-
vious reports on other strains and species of staphylococci.
When S. aureus Wood 46 (6) was grown aerobically and
anaerobically at 378C, the culture density achieved was nine
times as high in the presence of oxygen. Cultures of Staph-
ylococcus simulans biovar staphylolyticus grown aerobi-
cally at 378C achieved approximately 17-fold greater cell
density than did anaerobic cultures (7). The different ratios
are probably due in part to other growth conditions as well
as to strain differences.
J. Food Prot., Vol. 65, No. 1
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The strain used in this study has been implicated in
food poisoning (unlike the strains used in the other studies)
and therefore may be more relevant to food safety. From a
food safety perspective, the results of this study imply that
anaerobic conditions are not an effective barrier to S. au-
reus growth in food. There was only 2- to 3-fold S. aureus
growth reduction in anaerobic cultures. Moreover, signiŽ -
cant growth was observed at low temperatures with anaer-
obic conditions imposed even though the combination of
anaerobiasis and lower temperatures resulted in greater
growth rate reduction.
A model for S. aureus growth at various tempera-
tures in aerobic and anaerobic environments. We used
the data from Figure 4 to develop a linear model for S.
aureus aerobic and anaerobic growth as a function of the
incubation temperature. To calculate the average generation
time at various temperatures, we used the time needed to
double the optical density (at an optical density of approx-
imately 0.3) as the generation time. A regression analysis
of the growth as dependent on the temperature was per-
formed by a Ž rst-order regression analysis, Y 5 a 1 bX,
where Y is the log generation time in minutes and X is the
temperature log in degrees Celsius. The plot for this anal-
ysis is shown in Figure 5.
For aerobic growth, the regression equation is Y 5 7.08
2 3.55X with a regression coefŽ cient of r2 5 0.99. For
J. Food Prot., Vol. 65, No. 1 S. AUREUS GROWTH AND ENTEROTOXIN A PRODUCTION
FIGURE 5. A model for S. aureus growth at various temperatures
in aerobic and anaerobic environments; linear regression analy-
sis of temperature log versus log of generation time: v, aerobic
cultures; V, anaerobic cultures.
anaerobic growth, the regression equation is Y 5 6.23 2
2.794 with a regression coefŽ cient of r2 5 0.99. To evaluate
the validity of these equations, the generation time calcu-
lated from the equations was compared with the actual mea-
sured generation time of another set of measurements at 17,
22, 34, and 35.58C for both aerobic growth (Fig. 6A) and
anaerobic growth (Fig. 6B). With the calculated values plot-
ted against the measured values, the two equations (Fig.
203
6C) were used for both aerobic and anaerobic conditions.
The regression coefŽ cient of both plots was high (r2 5
0.98). This Ž nding suggests that the model can accurately
predict S. aureus generation time in the temperature range
tested. However, the deviation of the calculated generation
time from the actual measured generation time is greater
for 178C incubation than for higher temperatures. Because
of the long generation time of the cells at 178C incubation,
such a deviation (approximately third doubling time) is
probably not signiŽ cant.
Although a comprehensive model of the combined ef-
fects of temperature, pH, NaCl, sodium nitrite, and aeration
on staphylococcal growth has been developed (4), for food
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establishments and nonmanufacturing facilities, temperature
is the primary parameter that is varied. Therefore, we have
provided a less comprehensive linear model of staphylo-
coccal growth, one that depends on temperature only. This
simpliŽ ed model should be more useful in nonmanufactur-
ing settings that typically control only temperature in order
to maintain food products.
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