Med. Weter. 2015, 71 (6), 341-344
341
Praca oryginalna
Original paper
Staphylococcus aureus is among the top five
patho-gens responsible for acquired foodborne illnesses in
Europe (7). Staphylococcal food poisoning (SFP)
develops after ingestion of products containing
staphy-lococcal enterotoxins (SEs) and is manifested by
vom-iting, frequently accompanied by gastroenteritis. SEs
are a group of heat-stable, pepsin-resistant exotoxins
belonging to a large family of pyrogenic toxin super-
antigens (PTSAgs) encoded on phage, pathogenicity
islands, chromosome, or plasmids (1, 5). In addition to
the initially discovered SEA to SEE, known as
classi-cal SEs, a number of new enterotoxins have later been
described (28, 34). To date, twenty-four members of
the SE family have been identified. Some of the new
toxins, namely SEG, SEH, SEI, and SER, have been
shown to have emetic effect (25, 28, 32). Emetic effect
of another new toxin, SElP, has been demonstrated in
a small rodent, but not confirmed in a primate model.
Thus, despite its potential emetic activity, SElP still
cannot be classified as a true SE (27). It is estimated
that 5-10% of SFP cases in which none of SEA-SEE
enterotoxins were detected can be attributed to other
emetic SEs (11).
Genetic background can affect the repertoire of
mobile genetic elements in S. aureus (23). Some
S. aureus genotypes can occur in both animals and
humans (12, 24). However, it seems that the
associa-tion between certain enterotoxin genes and specific
staphylococcal clones may differ in S. aureus isolates
of human and animal origin (12, 33).
The aim of this study was to determine the incidence
of genes encoding emetic SEs in S. aureus isolates from
pork and pigs, referring the enterotoxigenic potential
of S. aureus to its genetic background.
Material and methods
Bacterial isolates. Ninety S. aureus isolates obtained by
Krupa et al. (18, 19) from pork (45 isolates) and pigs (45
Distribution of enterotoxin genes
in Staphylococcus aureus isolates from pork and pigs
JAROSŁAW BYSTROŃ, MAGDALENA PODKOWIK, JACEK BANIA,
PAWEŁ KRUPA, JUSTYNA SCHUBERT
Department of Food Hygiene and Consumer Health Protection, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, PolandReceived 20.01.2015 Accepted 28.01.2015
Bystroń J., Podkowik M., Bania J., Krupa P., Schubert J.
Distribution of enterotoxin genes in Staphylococcus aureus isolates from pork and pigs
Summary
Aim of the study: The study was conducted to determine the incidence of genes encoding emetic staphylococcal
enterotoxins (SEs) in S. aureus isolates from pork and pigs, and to demonstrate the connection between the
enterotoxigenic potential of S. aureus and its genetic background.
Materials and methods: S. aureus isolates from pork (45 isolates) and pigs (45 isolates), representing various
clonal complexes, were tested for the presence of emetic SEs genes.
Results and discussion: Thirty-four of the 45 S. aureus isolates (75%) derived from pork were shown to
harbor genes encoding emetic SEs. Among 45 pig-derived S. aureus isolates, SE genes were detected in 28
isolates (62%). Fifty-five percent of potentially enterotoxigenic staphylococci carried genes encoding classical
toxins (SEA-SEE), whereas 28 isolates (45%) harbored exclusively genes encoding new emetic SEs. The most
prevalent (82%) classical enterotoxin gene was seb, whereas seg and sei genes dominated (82%) among isolates
harboring genes encoding other emetic toxins. Seventeen of 23 S. aureus isolates assigned to the CC15 clonal
complex were found to harbor the seb gene. Ten of 15 CC7 isolates contained the selp gene. Isolates harboring
seg and sei genes dominated in CC30 (81%) and CC9 clones (76%). Four isolates assigned to CC398 were
shown to harbor enterotoxin genes, such as seb, sed, seg, sei, and ser. Our results indicate a high incidence
of enterotoxigenic S. aureus isolates harboring genes encoding other emetic SEs in pork and pigs. In most of
the pig- and pork-derived isolates studied here, genotype-enterotoxin association was similar to that known
from human S. aureus isolates. This is the first report on SE genes in S. aureus CC398 genetic background in
Poland, and probably also in Europe.
Med. Weter. 2015, 71 (6), 341-344
342
isolates) were used in this study. The animal-derived strains
were isolated from pigs’ nasal swabs in slaughterhouses, and
the meat-derived isolates were obtained from retail pork in
the Lower Silesia region (Poland). The isolates were
iden-tified as S. aureus on the basis of their ability to coagulate
rabbit plasma and clumping-factor production. All isolates
were screened by PCR with primers for the S. aureus
spe-cific nuc gene as described by Martin et al. (22). Reference
S. aureus strain ATCC 29213 served as a control. spa
geno-types of all the isolates and their assignment to clonal
com-plexes (CC) were determined previously (18, 19) according
to the method described by Krupa et al. (17).
Preparation of bacterial DNA. Two milliliters of a
bac-terial cell suspension from an overnight culture grown in
brain-heart infusion broth were centrifuged for 5 min at
12 000 × g, and suspended in 100 µl of 100 mM Tris-HCl
buffer, pH 7.4, containing 10 µg of lysostaphin
(Sigma-Aldrich, Poznan, Poland). After 30 min of incubation at
37°C, 10 µl of 10% SDS was added, and the sample was
incubated for another 30 min at 37°C. Two hundred µl of
5 M guanidine hydrochloride was added, and the sample
was mixed by vortexing and incubated at room temperature
for 10 min. DNA was extracted with phenol and chloroform,
ethanol-precipitated, and dissolved in water.
Detection of enterotoxin genes by PCR. The
detec-tion of genes coding for the enterotoxins SEA to SEE
was performed by the method described by Sharma et al.
(31). The primers for seg, seh, sei, selp, and ser detection
were designed on the basis of the alignment of published
sequences, as described in our previous works (2, 3). The
ser gene was detected with the following primers: SER-for
GTGCTAAACCAGATCCA, SER-rev
CTAGCTCTTG-TACCGTA.
Enterotoxin genes were detected by four PCR reaction
mixtures. The first contained primers for sea, sec, and see.
The second contained primers for seb and sed, the third
contained primers for seg, seh, sei, and selp, and the fourth
contained primers for ser. Five enterotoxigenic reference
strains were used as positive controls: FRI137 (sec, seg,
seh, sei), FRI913 (sea, sec, see), CCM5757 (seb), FRI
1151m (sed, ser) (34), and A900322 (seg, sei, selp). The
PCR was performed in a total volume of 25 µl. In each
case, the reaction mixture contained 1 × polymerase buffer
with 50 mM KCl, 10 mM Tris-HCl, 4 mM MgCl
2, 0.2 mM
of each dNTP, 30 pmole of each primer (Institute of
Bio-chemistry and Biophysics, Warsaw, Poland), 1 µl of DNA
solution, and 1 U of Taq DNA polymerase (Fermentas,
Vilnius, Lithuania). Thirty-five cycles of 95°C for 30 s,
52°C for 30 s, and 72°C for 1.5 min were performed with
a MJMini thermal cycler (BioRad, Hercules, USA). Each
PCR was run with a mix of DNA from the corresponding
reference strains as positive controls. Ten-µl aliquots of
PCR products were resolved on 2% agarose gel at 100 V
and documented with a GelDoc XR documentation system
(BioRad, Hercules, USA).
Results and discussion
Thirty-four of the 45 S. aureus isolates (75%)
derived from pork were shown to harbor genes
encod-ing emetic SEs. In 23 isolates, sea-see genes were
detected, whereas 11 isolates contained other SE genes.
Specifically, the seb gene was found in 20 isolates,
selp in 8 isolates, seg and sei in 5, sec in 4, and seh
in 2 isolates. Ten isolates harbored two or three SE
genes (Tab. 1).
Among 45 pig-derived S. aureus isolates, SE genes
were detected in 28 isolates (62%). In 11 isolates,
sea-see genes were found, and 17 isolates contained
other SE genes. In 18 isolates, seg and sei genes were
found, seb was detected in 8, selp in 6, sec in 3, sed
and ser in 1 isolate. Nineteen isolates harbored two or
three SE genes (Tab. 1).
Seventeen of 23 S. aureus isolates assigned to the
CC15 clonal complex were found to harbor the seb
gene. Ten of 15 CC7 isolates included the selp gene.
Tab. 1. Incidence of staphylococcal enterotoxin genes (SEs) in pig- and pork-derived S. aureus isolates representing various
clonal complexes
Distribution of SEs in S. aureus isolates
Pigs
(number of enterotoxigenic isolates/number of isolates) (number of enterotoxigenic isolates/number of isolates)Pork (number of isolates)Clonal complex no isolates (0/0) seb (1/3); seh (1/3); seb + seh (1/3) CC1 (3)
selp (4/7) selp (6/8) CC7 (15)
no isolates (0/0) sec (1/3); seb + selp (1/3) CC8 (3)
seb (1/11); seg + sei (2/11); seb + seg + sei (4/11); sec + seg + sei (2/11) seg + sei (1/2); selp + seg + sei (1/2) CC9 (13) no isolates (0/0) sec + selp (1/1) CC12 (1)
selp (2/4) seb (15/19); seb + sec (2/19) CC15 (23)
no isolates (0/0) seg + sei (1/1) CC25 (1)
seg + sei (8/10) seg + sei (1/1) CC30 (11)
sec (1/1) sec + seg + sei (1/1) CC45 (2)
no isolates (0/0) not detected (0/1) CC97 (1) no isolates (0/0) not detected (0/1) CC101 (1)
seb (1/12); seb + sed + ser (1/12); seb + seg + sei (1/12); seg + sei (1/12) not detected (0/4) CC398 (16) Total: 28/45 Total: 34/45 Total: 90
Med. Weter. 2015, 71 (6), 341-344
343
Isolates harboring seg and sei genes dominated in
CC30 (81%) and CC9 clones (76%). Four isolates
assigned to CC398 were shown to harbor enterotoxin
genes, such as seb, sed, seg, sei, and ser (Tab. 1).
Relatively little is known on the enterotoxigenic
potential of livestock-associated S. aureus. In this
study, 69% of 90 S. aureus isolates examined
har-bored genes encoding emetic enterotoxins. Fifty-five
percent of potentially enterotoxigenic staphylococci
carried genes encoding classical toxins (SEA-SEE),
whereas 28 isolates (45%) harbored exclusively genes
encoding new emetic SEs. The most prevalent (82%)
classical enterotoxin gene was seb, whereas seg and
sei genes dominated (82%) among isolates harboring
genes encoding other emetic toxins. Similarly, among
enterotoxigenic S. aureus from pigs in Switzerland,
isolates containing seg and sei genes were dominant
(63%), whereas classical toxin genes, represented by
the sec gene, were detected in only 2% of the isolates
(26). The seg and sei genes were also the most
preva-lent enterotoxin genes among S. aureus isolates from
retail meat, including pork, in Korea and the United
States (14, 29). These two genes are usually detected
together in S. aureus (15). As found by Jarraud et al.
(15), seg and sei, together with selm, seln, and selo, are
linked in an operon called the enterotoxin gene cluster
(egc). Evidence for SEG and SEI expression is indirect
only, so their role in SFP remains unclear (13).
Certain S. aureus genotypes were shown to be
asso-ciated with a specific enterotoxin gene repertoire (23).
In most of the pig and pork-derived isolates studied
here, genotype/enterotoxin association was similar to
that known from human S. aureus isolates. This applies
especially to seb and seh genes, which were frequently
found in CC1 (20), to selp, which was found in CC7,
as well as to sec and selp, prevailing in CC12 (12).
The seg and sei genes, belonging to egc, have already
been associated with CC9, CC30, CC25, and CC45
background in human, food, and bovine S. aureus
isolates (8, 12, 30). According to our data, seg and
sei also dominate in the abovementioned genotypes
of pig-associated S. aureus. The lack of enterotoxin
genes is a characteristic trait of bovine CC97 and
CC101 S. aureus isolates (30). According to our data,
S. aureus CC97 and CC101 from pigs can also
con-stitute unfavorable background for SE incorporation.
Relatively little is known about the SE gene content
in the livestock-associated CC398 lineage. Studies
on European isolates belonging to CC398 report the
absence of SE genes from this genetic background (3,
8-10, 16, 21, 24, 30). On the other hand, 33% of
pig-associated S. aureus population studied here was found
to be enterotoxigenic. Liu et al. (20) recently reported
on the incidence of SEs in S. aureus CC398 of human
origin in China. The seb, seg and sei genes detected
in Chinese CC398 human isolates were also found in
our CC398 pig-associated isolates.
According to the European legislation on food
safety, SEA-SEE are the only SEs routinely detected
in food (6). Our results indicate a high incidence of
enterotoxigenic S. aureus isolates harboring genes
encoding other emetic SEs in pork and pigs, which
implies the need for new methods of tracking currently
underestimated food hazards. The present study is the
first report on SE genes in S. aureus CC398 genetic
background in Poland, and probably also in Europe.
This indicates that a typical animal-associated S. aureus
clone regarded so far as neutral for food safety may in
fact pose a potential risk for consumers.
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Corresponding author: dr hab. Jarosław Bystroń, prof. nadzw., ul. C. K. Norwida 31, 50-375 Wrocław; e-mail: jaroslaw.bystron@up.wroc.pl
