Short Communications

High Incidence of Enterotoxin D Producing Staphylococcus
spp. in Brazilian Cow’s Raw Milk and Its Relation
with Coagulase and Thermonuclease Enzymes

Ana Maria Oliveira,1 Carlos Roberto Padovani,2 Norma Teruko Nagô Miya,1

Anderson S. Sant’Ana,3 and José Luiz Pereira1

Abstract

In this study, the enterotoxigenic potential of Staphylococcus strains (n¼ 574) isolated from raw milk samples
(n¼ 140) was determined for their capacity to produce staphylococcal enterotoxins. In addition, the relationship
between the presence of enterotoxins, coagulase, and thermonuclease (Tnase) was assessed. The results showed
that 19% of Staphylococcus was enterotoxigenic, being able to produce at least one of the staphylococcal en-
terotoxins (A, B, C, and D). Most of the strains were able to produce enterotoxin D (68.8%), whereas 12.8% of the
Staphylococcus strains were able to produce staphylococcal enterotoxin A. Besides, the production of more than
one type of enterotoxins by the same strain was observed. Tnase was considered the best marker for en-
terotoxigenic potential of isolates, although some of them were negative for coagulase and Tnase but positive for
enterotoxin production. Therefore, either the use of Tnase to assess Staphylococcus enterotoxigenic potential or
the use of simple and easy screening tests for enterotoxin production should receive more attention when
evaluating the pathogenic potential of foodborne Staphylococcus strains. Due to the association of both coagulase
positive Staphylococcus and coagulase negative Staphylococcus with foodborne disease outbreaks, regulators and
industries should pay more attention to enterotoxigenic Staphylococcus rather than focusing only on S. aureus or
coagulase positive Staphylococcus. Finally, data found here suggest a high risk of staphylococcal intoxication with
the consumption of raw milk or dairy products made from raw milk.

Introduction

Staphylococcus spp. are widespread microorganisms
that play an important role in foods as beneficial micro-

organisms (Irlinger, 2008; Zell et al., 2008; Coton et al., 2009) or
as agents of foodborne diseases (Miwa et al., 2001; Do Carmo
et al., 2002, 2003; Colombari et al., 2007; López et al., 2008).
Foodborne diseases associated with enterotoxigenic Staphy-
lococcus are caused by the consumption of foods contaminated
with preformed enterotoxins (Le Loir et al., 2003). Among
staphylococcal enterotoxins, type-A enterotoxin is responsible
for almost 80% of outbreaks (Balaban and Rasooly, 2000).

The ability of Staphylococcus to produce coagulase is used to
indicate enterotoxigenic potential of isolates, which are called
coagulase positive Staphylococcus (CPS). Due to this, they have
received more attention by both governments and industries
when establishing regulations, microbiological specifications,

or methods. Despite this, the ability of coagulase negative
Staphylococcus (CNS) to produce enterotoxins (Vernozy-
Rozand et al., 1996; Udo et al., 1999; Zell et al., 2008) and their
association with foodborne disease outbreaks have been re-
ported (Do Carmo et al., 2002; Veras et al., 2008). The presence
of CNS in foods is of concern, as they are commonly found in
raw material (e.g., as milk) and may be used as starter cultures
for production of fermented foods (Zell et al., 2008). Since
analytical methodology assumes that enterotoxigenic Sta-
phylococcus must produce coagulase (Lancette and Bennett,
2001), the presence of CNS is overlooked and, thus, implica-
tions for food safety may arise. Therefore, testing the potential
of Staphylococcus strains to produce enterotoxins using easy
and inexpensive methods (Robbins et al., 1974) may be a better
choice so as to report on the enterotoxigenic potential of Sta-
phylococcus strains. This study aimed at determining the en-
terotoxigenic potential of Staphylococcus strains isolated from

1Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo, Brazil.
2Department of Statistics, Institute of Biosciences, São Paulo State University, Botucatu, São Paulo, Brazil.
3Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

FOODBORNE PATHOGENS AND DISEASE
Volume 8, Number 1, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089=fpd.2010.0590

159



Brazilian raw milk and estimating the relationship between
production of enterotoxins and coagulase and thermo-
nuclease (Tnase) enzymes.

Materials and Methods

Raw milk sampling

Raw milk samples (200 mL) of nine different and randomly
selected milk producers and a pool of them were collected
during 10 months. These samples (n¼ 140) were sent by milk
producers to a cooperative dairy plant located in the city of
Campinas, State of São Paulo (Brazil), for milk and dairy
production. Samples were collected under aseptic conditions,
transported, and maintained at 48C. Microbiological analysis
was performed at the same day of sampling.

Enumeration and identification of Staphylococcus spp.

The enumeration and identification of Staphylococcus spp.
in raw milk samples were performed as proposed by Lancette
and Bennett (2001). An aliquot of samples (10 mL) was ho-
mogenized in 90 mL of 0.1% sterile peptone water. Decimal
dilutions were prepared in tubes containing 9 mL of this dil-
uent and inoculated onto Baird Parker agar (Oxoid, Basing-
stoke, United Kingdom) plates. The inoculum was spread
over the surface of the agar using sterile rods. Plates were kept
in an upright position until liquid was absorbed. Baird Parker
agar plates were inverted and incubated at 378C for 48 h.
Characteristic colonies of Staphylococcus spp. (circular, smooth
or rough appearance, convex, gray-black surrounded by
opaque and clear zones or not) were counted. About 5–10
colonies of each sample were picked up for confirmation.
Confirmation at genera level was performed by using Gram’s
staining, production of catalase, coagulase, heat stable nu-
clease (Tnase), and anaerobic utilization of mannitol tests
(Lancette and Bennett, 2001). Coagulase was performed using
the tube method as described by Lancette and Bennett (2001),
whereas Tnase production was assessed by the plate tech-
nique with toluidine blue-deoxyribonucleic acid agar (Lachi-
ca, 1976). Anaerobic fermentation of mannitol was used when
isolates were negative for coagulase and Tnase tests to dif-
ferentiate Staphylococcus spp. from Micrococcus spp. Since

Micrococcus is a strict aerobe, mannitol fermentation indicated
that an isolate was within Staphylococcus genus (Schleifer,
1986). An isolate was considered within Staphylococcus genus
when presented as gram-positive cocci in grape-like clusters,
catalase, and mannitol fermentation positive. A 3þ or 4þ clot
formation was considered as a positive reaction for Staphylo-
coccus, whereas isolates presenting 1þ and 2þ clot types were
only considered positive when Tnase test was also positive.
The number of isolates that presented positive and negative
results in coagulase and Tnase tests was recorded. Isolates
presenting positive or negative reactions for coagulase and
positive reaction for Tnase were investigated for their ability
in producing enterotoxins.

Enterotoxin production and detection

For enterotoxin production, each of the Staphylococcus iso-
lates were inoculated in brain–heart infusion (BHI) broth
(Difco, Sparks, NV). Tubes were incubated overnight at 378C.
BHI Agar plates were covered with membrane disks made
from Spectra=membrane dialysis tubing, 6000–8000, 100 mm
flat width (Thomas Scientific, Philadelphia, PA). Then, 0.5 mL
of each isolate grown in BHI broth was inoculated in mem-
branes, and the plates were incubated at 378C for 24 h.
Membranes were washed twice with 1.5 and 1 mL, respec-
tively, of 0.02 M NaHPO4. Then, cultures were centrifuged
and culture supernatants were used for enterotoxin detection
(Robbins et al., 1974; Pereira et al., 1996).

For enterotoxin detection, the optimum-sensitivity plate
(OSP) method was used (Robbins et al., 1974). Noble agar
(1.2%) prepared in 0.02 M phosphate-buffered saline, pH 7.4
was put in 50 mm diameter Petri dishes. Then, seven wells (five
with 8.3 mm and two with 6.7 mm diameter) were cut in the
agar. The antiserum, control enterotoxin, and samples were
placed in the central, in the two smaller and in the four larger
outer wells, respectively. Petri dishes were closed and stored in
a humidified box at 378C for 18 h. The union of the precipita-
tion line produced by isolate fluids with those formed by
control indicated a positive reaction. To detect low-enterotoxin-
producing strains, culture supernatant fluids were concen-
trated 10 times. A, B, C, and D Staphylococcal enterotoxins,
together with their respective antiserum, were gently obtained

Table 1. Percentage of Enterotoxigenic Staphylococcus spp. in Raw Milk

Enterotoxigenicity of Staphylococcus strains (%)

Milk producers Positive Negative Total number of isolates tested

Pool 15.2a,b,A 84.8a,b,B 66
P2 18.4a,b,A 81.6b,c,B 87
P3 11.1a,b,A 88.9a,b,B 45
P4 15.6a,b,A 88.4a,b,B 64
P5 3.3c,A 96.7a,B 61
P6 24.1a,b,A 75.9b,c,B 54
P7 29.8a,b,A 70.2b,c,B 47
P8 14.6a,b,A 85.4a,b,B 48
P9 17.9a,b,A 82.1a,b,c,B 39
P10 39.7a,A 60.3c,B 63
Mean 19 81 574

Values in the same line with different capital letters indicate significant difference (95%) between enterotoxigenic and nonenterotoxigenic
Staphylococcus strains for the same producer. Values in the same column with different small letters indicate significant difference (95%)
between enterotoxigenic and nonenterotoxigenic Staphylococcus strains among producers.

160 OLIVEIRA ET AL.



from M.S. Bergdoll (Food Research Institute, University of
Wisconsin-Madison). Different dishes were used for testing
enterotoxin production by raw milk Staphylococcus isolates.

Statistical analysis

Goodman’s test was used for contrasts among multinomial
proportions at 5% significance level (Sheskin, 2000).

Results and Discussion

In Brazil, Salmonella spp. is the first agent causing food-
borne diseases, whereas enterotoxigenic Staphylococcus is the
second most important microorganism associated with these
diseases. Staphylococcus are blamed for more than 572 out-
breaks from 1999 to 2007 (ANVISA, 2008). Several outbreaks
caused by these microorganisms have been reported in the
literature (Do Carmo et al., 2002, 2003, Colombari et al., 2007;
Veras et al., 2008), and some of them are linked to dairy
products.

In this study, all samples presented presumptive Staphylo-
coccus colonies, and counts of these microorganisms ranged
around 8.9�103–1.5�105 colony forming units (CFU)=mL,
which may be indicative of poor hygiene during milking or in
temperature control during transportation, as the milk came
from healthy cows. From 140 samples, 574 Staphylococcus
strains were isolated and tested for enterotoxins production.
Despite the significant difference between some milk pro-
ducers (from 11.1% to 39.7%) according to Goodman’s statis-
tical test ( p< 0.05), it is possible to see in Table 1 that the
average rate for the incidence of enterotoxigenic Staphylococcus
was 19% (n¼ 109). Although the percentage of enterotoxigenic
strains cannot be considered high, it must be observed with
concern by milk farmers and dairy processing plants. For milk

farmers, the results found here indicate that although pro-
grams on mastitis control may be successful, more care should
be taken with hygiene and temperature control between
milking and milk being received at the dairy plant for pro-
cessing. Counts of Staphylococcus aureus higher than 106

CFU=g or mL in foods are reported as the critical level for these
microorganisms to produce enterotoxins and to cause food-
borne disease outbreaks (Balaban and Rasooly, 2000; Le Loir
et al., 2003). However, even low counts such as 103 CFU=g
reached by S. aureus may be enough for this microorganism to
produce enough amount of enterotoxin (1 ng=g) to cause in-
toxication (Pereira et al., 1991). This reinforces the need of dairy
processing plants working together with milk farmers so as to
improve the microbiological quality of raw milk. Besides being
a source of contamination by Staphylococcus in the processing
environment, the presence of enterotoxins in raw milk samples
may lead to the production of high-risk dairy products. This is
because staphylococcal enterotoxins are highly stable to the
steps used in dairy processing, mainly milk pasteurization
(Modi et al., 1990; Schwabe et al., 1990).

According to Table 2, Staphylococcus spp. isolated from
Brazilian raw milk samples were mainly producers of type
D enterotoxin (68.8%), with less than 10% of the strains pro-
ducing two different types of enterotoxins. The percentage of
isolates producing enterotoxin type A was low. Currently,
more than 20 types of staphylococcal enterotoxins are known
(Balaban and Rasooly, 2000; Pereira et al., 1991; Munson et al.,
1998; Orwin et al., 2001; Omoe et al., 2005; Thomas et al., 2006;
Ono et al., 2008). However, enterotoxin A is the most com-
monly staphylococcal enterotoxin associated with foodborne
outbreaks, followed by enterotoxin D (Balaban and Rasooly,
2000). Data obtained here indicated that enterotoxin D may
play an important role in staphylococcal food poisoning in

Table 2. Types of Enterotoxins Produced by 109 Raw Milk Staphylococcus spp. Isolates

Types of enterotoxins produced by Staphylococcus spp. isolated from raw cow milk (%)

A B C D A-B A-D B-D

12.8 7.3 2.8 68.8 3.7 ND 4.6

ND, nondetected.

Table 3. Correlation Between Enterotoxin Production, Coagulase, and Thermonuclease

by Raw Milk Staphylococcus spp. Isolates

Producer
Tnase (þ) Coag

(þ) (%)
Tnase (�) Coag

(�) (%)
Tnase (þ) Coag

(�) (%)
Tnase (�) Coag

(þ) (%)
Number of

cultures tested

Pool 20c,A 30a,A 40b,c,A 10A 10
P2 87.5a,b,A 0.0 12.5c,B 0.0 16
P3 20c,B 0.0 80a,A 0.0 05
P4 100 0.0 0.0 0.0 10
P5 100 0.0 0.0 0.0 02
P6 38.5b,c,A 0.0 61.5a,A 0.0 13
P7 35.7b,c,A 0.0 64.3a,A 0.0 14
P8 42.8b,c,A 28.6a,A 28.6b,c,A 0.0 07
P9 85.7a,b,A 0.0 14.3b,c,B 0.0 07
P10 92a,b,A 0.0 8.0c,B 0.0 25

Values in the same line with different capital letters indicate significant difference (95%) regarding coagulase and Tnase production by
enterotoxigenic Staphylococcus strains for the same producer. Values in the same column with different small letters indicate significant
difference (95%) regarding coagulase and Tnase production by enterotoxigenic Staphylococcus strains among producers.

Tnase, thermonuclease; Coag, coagulase.

ENTEROTOXIN D PRODUCING STAPHYLOCOCCUS IN COW’S RAW MILK 161



Brazil, as this enterotoxin and type A enterotoxin have been
commonly associated with outbreaks in which Staphylococcus
strains have been implicated (Do Carmo et al., 2002, 2003; Veras
et al., 2008). However, which type of staphylococcal entero-
toxins is more associated with foodborne disease outbreak
seems not to be relevant. This is because staphylococcal en-
terotoxins have very similar structure and function (Balaban
and Rasooly, 2000) and will cause known symptoms if they are
found in foods in enough amount to cause food poisoning.

OSP method has been chosen to screen staphylococcal en-
terotoxins, as it requires an easy sample preparation and
setup, simple reagents, and has a sensitivity of 0.5 mg=mL (Su
and Wong, 1997). Table 3 shows the correlation between en-
terotoxigenic Staphylococcus strains and their ability to pro-
duce coagulase and Tnase. Pools P3, P6, and P7 presented
higher percentages of isolates within Tnase (þ) and coagulase
(�) group, whereas a significant difference between entero-
toxin and enzymes production was only found in P3
( p< 0.05). It can be noticed that in pool and P8 milk pro-
ducers, up to 30% of isolates were enterotoxigenic and even
presented negative results for Tnase and coagulase tests
( p> 0.05). Isolates from P4, P5, P9, and P10 milk producers
presented the higher percentages of enterotoxin production
when Tnase and coagulase were positive ( p< 0.05). Among
all milk producers, only one strain (10%) of 10 tested in pool,
presented enterotoxigenic potential and Tnase (�) and coag-
ulase (þ). Staphylococcal isolates from pool and P3 milk
producers yielded the lower percentages of correlation (20%)
between enterotoxin and coagulase=Tnase production
( p< 0.05). This research found that a high percentage of en-
terotoxigenic Staphylococcus isolates were coagulase negative.
Danielsson and Hellberg (1977) also reported that CNS iso-
lated from food handlers produced SE. Additionally, data
indicated that the Tnase test is more faithful to assess patho-
genic potential of foodborne Staphylococcus. So, either the use
of Tnase to assess Staphylococcus enterotoxigenic potential or
the use of simple and easy screening tests for enterotoxin
production such as OSP (Robbins et al., 1974) could be con-
sidered. Due to the association of both CPS and CNS with
foodborne disease outbreaks, regulations and food industries
should pay more attention to enterotoxigenic Staphylococcus
rather than focusing only on S. aureus or CPS. Finally, data
found here suggest a high risk of staphylococcal intoxication
by raw milk or dairy products made from raw milk due to the
enterotoxigenic potential of the strains isolated.

Acknowledgments

The authors acknowledge CNPq (Conselho Nacional
de Desenvolvimento Cientı́fico e Tecnológico), Capes (Co-
ordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior),
and FAPESP (Fundação de Amparo à Pesquisa do Estado de
São Paulo) for the financial support to this study.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
José Luiz Pereira, Ph.D.

Deparment of Food Science
Faculty of Food Engineering

University of Campinas
Rua Monteiro Lobato 80

Caixa Postal 6121
Campinas, SP 13081-970

Brazil

E-mail: pereira@fea.unicamp.br

ENTEROTOXIN D PRODUCING STAPHYLOCOCCUS IN COW’S RAW MILK 163