Full text of DOI:10.1007/PL00011281

Eur J Clin Microbiol Infect Dis (2001) 20:407–409
Q Springer-Verlag 2001
Concise Article
Survey of Staphylococcal Enterotoxin Genes,
Exfoliative Toxin Genes, and Toxic Shock Syndrome
Toxin 1 Gene in Non-Staphylococcus aureus Species
K. Becker, G. Haverkämper, C. von Eiff, R. Roth, G. Peters
Abstract The aim of this study was to analyze the pres- aureus [1]. In contrast to the amount of literature avail-
ence of staphylococcal exotoxin genes in staphylococci able for Staphylococcus aureus, only very few reports
other than Staphylococcus aureus. Whereas for Staphy- have been published concerning the detection of these
lococcus aureus a large spectrum of different exotoxins exotoxins in coagulase-negative staphylococci (CNS) or
responsible for toxin-mediated diseases has been coagulase-positive/-variable non-Staphylococcus aureus
described, only few and conflicting data are available staphylococci (NSAS) [2–7]. Because of the conflicting
regarding toxin production in all other staphylococcal results of these reports, there is an obvious need for
species. A collection of clinical non-Staphylococcus systematic screening for toxin production in CNS and
aureus staphylococcal isolates were systematically other NSAS. Furthermore, previous studies mainly
screened for the presence of genes for toxic shock used immunological methods and did not search for the
syndrome toxin 1, exfoliative toxins, and classical ente- presence of the respective genes.
rotoxins by using multiplex polymerase chain reaction
enzyme immunoassays. In a total of 461 isolates of 18 In this study, a large group of different CNS and coagu-
different (sub)species derived from clinical specimens, lase-positive/-variable (Staphylococcus hyicus, Staphy-
no toxin gene sequences were amplified. The results lococcus intermedius, Staphylococcus schleiferi subsp.
indicate that the occurrence of toxin genes involved in coagulans) and/or clumping factor-positive/-variable
staphylococcal toxin-mediated diseases is at least very NSAS (Staphylococcus intermedius, Staphylococcus
rare in human-derived isolates of non-Staphylococcus lugdunensis, Staphylococcus schleiferi subsp. schleiferi)
aureus species.
from clinical specimens were screened for the presence
of classical enterotoxin genes (sea, seb, sec1–3, sed, see),
exfoliative genes (eta, etb), and the TSST-1 gene (tst) by
two multiplex polymerase chain reaction enzyme
immunoassays (PCR-EIAs) [8]. In order to detect
Introduction
The production of staphylococcal enterotoxins A–E possible differences between human CNS strains from
(SEA, SEB, SEC, SED, SEE), exfoliative toxins A and the physiological skin flora and CNS strains involved in
B (ETA, ETB), and toxic shock syndrome toxin 1 significant clinical infections, e.g. bacteremia, isolates
(TSST-1), responsible for toxin-mediated staphylo- from a German multicenter study were included [9].
coccal diseases such as food poisoning, staphylococcal
scalded skin syndrome, and toxic shock syndrome, is
well-documented for defined strains of Staphylococcus
Materials and Methods
A total of 461 clinical isolates of 18 staphylococcal non-Staphylo-
coccus aureus (sub)species were included in the study (Table 1).
They were taken from blood (np336) or other clinical specimens
(skin, wounds, respiratory tract, urogenital tract, venous
catheters; np125) collected prospectively in one center (Univer-
sity of Münster). Furthermore, 71 blood culture isolates from a
nationwide German multicenter study (21 tertiary-care hospitals)
on the prevalence of antibiotic resistance in staphylococci from
patients with bacteremia [9] were tested. In that multicenter
study, CNS were included only if they were recovered from at
least two separate sets of blood cultures. Only one isolate per
patient was included in this study.
K. Becker (Y), G. Haverkämper, C. von Eiff, R. Roth,
G. Peters
Institut für Medizinische Mikrobiologie,
Universitätsklinikum Münster
Domagkstraße 10, 48149 Münster, Germany
e-mail: kbecker6uni-muenster.de
Tel.: c49-251-8355360
Fax: c49-251-8355350

408
Table 1 Staphylococcal (sub)species included in the study, along
species distribution within the study corresponded
approximately to the frequency of occurrence of the
different CNS and other NSAS species in routine diag-
nostic laboratories, i.e. most of the strains studied were
Staphylococcus epidermidis, followed by Staphylo-
coccus haemolyticus and Staphylococcus hominis subsp.
hominis (Table 1). No genes of staphylococcal entero-
toxins (SEA–SEE), exfoliative toxins (ETA, ETB), or
TSST-1 were detected by the two multiplex PCR-EIAs
in the human-derived coagulase-negative and clumping
factor-negative strains, in the strains of coagulase-posi-
tive/-variable (sub)species, or in the strains of clumping
factor-positive/-variable (sub)species tested (Table 1).
with the source of clinical isolates
Staphylococcal taxon
No. of strains
Blood
Other
specimens
Total
culturesa
S. auricularis
S. capitis subsp. capitis
S. caprae
S. cohnii subsp. cohnii
S. cohnii subsp. urealyticus
S. epidermidis
S. haemolyticus
S. hominis subsp. hominis
S. hyicus
S. intermedius
S. lugdunensis
0
1
2
1
21
6
19 (2)
2
4
2
4
0
6
1
1
239 (56)
36
7
275
31
23
1
24 (8)
22 (4)
1
0
0
5
6
1
1
1
30
14
Regarding the clinical significance of the CNS isolates
tested, staphylococcal toxin genes were detected
neither in isolates derived from clinically presumed foci
of infection (e.g. catheter) nor in isolates from skin and
mucous membranes. The same finding holds true for
the strains from a German multicenter study in which
only CNS blood isolates considered etiologically rele-
vant were included.
25
8
S. saprophyticus subsp.
saprophyticus
S. schleiferi subsp. schleiferi
S. schleiferi subsp. coagulans
S. sciuri subsp. sciuri
S. simulans
0
0
6
1
6
1
3 (1)
7
10
11
15
8
2
9
S. warneri
8
7
S. xylosus
3
5
Total
336 (71)
125
461
CNS belong to the most frequently isolated bacteria in
the microbiological laboratory. However, only few and
contradictory data are available regarding their ability
to produce exotoxins [6, 12]. Crass and Bergdoll [2]
first described in 1986 the identification of CNS that
were positive for TSST-1 from patients with toxic shock
syndrome. Subsequently, several larger surveys in the
1980s and 1990s failed to detect this toxin in CNS of
human origin [13–15]. In contrast, detection of entero-
toxins and TSST-1 in at least 10 different CNS species
isolated from animal food products and other animal
sources has been reported [7]. Jaulhac et al. [16]
reported on two CNS strains that were hybridized by
DNA with an SEB and an SEC probe, respectively, but
for which the corresponding toxin could not be
detected. However, other studies failed to identify ente-
rotoxigenic CNS in animal-derived specimens [3, 4]. To
our knowledge, no data regarding exfoliative toxins are
published for CNS.
a Numbers in parentheses show the number of isolates that came
from a German multicenter study on the prevalence of anti-
biotic resistance in staphylococcal isolates from patients with
bacteremia [9]
Identification was based on growth characteristics on Columbia
agar with 5% (v/v) sheep blood at 37 7C and positive catalase
production. The clinical isolates were identified biochemically by
the automated ID 32 Staph system (bioMérieux, France). The
presence or absence of bound or free coagulase was confirmed by
latex slide agglutination for clumping factor, protein A, and
capsular polysaccharides (Pastorex Staph-Plus; Sanofi Diagnos-
tics Pasteur, France). In addition, an in-house slide coagulase test
using rabbit and human citrate plasma and a coagulase tube test
using rabbit plasma were performed. In the case of coagulase-
positive/-variable and clumping factor-positive/-variable NSAS
strains, a possible misidentification regarding Staphylococcus
aureus was excluded by PCRs for detection of the Staphylococcus
aureus-specific nuc gene, which codes for thermostable nuclease,
and the coagulase gene (coa); both PCR procedures were done
with primer pairs as described previously [10, 11].
In analogy to Staphylococcus aureus, an increased
pathogenicity of the predominantly animal-associated
coagulase-positive/-variable and/or clumping factor-
positive/-variable species in humans also may be
assumed. At least some of these staphylococcal species
appear to be significant human pathogens or were occa-
sionally isolated as putative etiological pathogens from
specimens of human origin. Hirooka et al. [5] described
25% of canine Staphylococcus intermedius isolates as
being enterotoxigenic. Adesiyun et al. [17] reported on
enterotoxigenic Staphylococcus hyicus isolates using a
monkey-feeding bioassay. In contrast to cited studies,
we included only isolates of human origin in this study.
This, and the limited number of these staphylococci
Procedures for culture and DNA isolation as well as the two
multiplex PCR-EIA systems – one designed to detect the staphy-
lococcal enterotoxin genes (sea, seb, sec1–3, sed, see) simulta-
neously and the other developed to detect the tst gene and the
exfoliative genes (eta, etb) concomitantly – were performed as
described previously [8]. Positive controls included DNA from
toxin-producing Staphylococcus aureus strains (SEA, KN543;
SEB, ATCC 14458; SEC, ATCC 19095; SED, ATCC 23235; SEE,
ATCC 27664; ETA, BM 10458; ETB, BM 10143; TSST-1, KN
813). In addition to standard PCR controls for contamination
events, Staphylococcus aureus Cowan 1 and Staphylococcus
epidermidis DSM 20044 were used as negative controls.
Results and Discussion
A total of 461 human isolates of 18 non-Staphylococcus studied, might explain the lack of positive results in
aureus staphylococcal (sub)species were included. The toxin gene amplification.

409
5. Hirooka EY, Muller EE, Freitas JC, Vicente E, Yoshimoto
Y, Bergdoll MS: Enterotoxigenicity of Staphylococcus inter-
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biology (1988) 7:185–191
6. Gemmell CG, Schumacher-Perdreau F: Extracellular toxins
and enzymes elaborated by coagulase-negative staphylococci.
In: Mardh PA, Schleifer KH (eds): Coagulase-negative
staphylococci. Almqvist and Wiksell, Stockholm (1986)
pp 809–827
A systematic survey for genes coding for staphylococcal
exotoxins is still lacking for CNS and other NSAS.
Almost all former studies have been based on immuno-
logical methods for measurement of toxin protein in
culture fluids of suspect strains or in food extracts.
These methods can be influenced by various factors,
resulting in nonspecific reactions. Hence, contrary to
previous investigations, the objective of our study was
to detect the presence of the genes, not the presence of
expressed toxin proteins.
7. Jay JM: Modern food microbiology. Chapman & Hall, New
York (1996) p 450
8. Becker K, Roth R, Peters G: Rapid and specific detection of
toxigenic Staphylococcus aureus: use of two multiplex PCR
enzyme immunoassays for amplification and hybridization of
staphylococcal enterotoxin genes, exfoliative toxin genes, and
toxic shock syndrome toxin 1 gene. Journal of Clinical Micro-
biology (1998) 36:2548–2553
9. von Eiff C, Reinert RR, Kresken M, Brauers J, Hafner D,
and Peters G for the Multicenter Study on Antibiotic Resist-
ance in Staphylococci and Other Gram-Positive Cocci Study
(MARS) Group: Nationwide German multicenter study on
prevalence of antibiotic resistance in staphylococcal blood-
stream isolates and comparative in vitro activities of quinu-
pristin-dalfopristin. Journal of Clinical Microbiology (2000)
38:2819–2823
10. Brakstad OG, Aasbakk K, Maeland JA: Detection of Staphy-
lococcus aureus by polymerase chain reaction amplification of
the nuc gene. Journal of Clinical Microbiology (1992)
30:1654–1660
11. Goh SH, Byrne SK, Zhang JL, Chow AW: Molecular typing
of Staphylococcus aureus on the basis of coagulase gene poly-
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Irrespective of the source of specimens tested, we did
not detect classical enterotoxin genes, exfoliative toxin
genes, or the TSST-1 gene in human-derived non-
Staphylococcus aureus staphylococcal isolates. Thus,
CNS and other NSAS strains harboring these toxin
genes appear to be uncommon in human specimens.
In conclusion, this is the first study in which a large
number of NSAS were systematically screened by
molecular methods for toxin genes. The results indicate
that the occurrence of staphylococcal enterotoxin,
exfoliative toxin, and TSST-1 genes in CNS and other
non-aureus staphylococci is at least very rare in isolates
of human origin.
Acknowledgements The authors are grateful to M. Brück, M.
Schulte, S. Deiwick, and B. Schuhen for excellent technical assist- 12. Bennett RW: Atypical toxigenic Staphylococcus and non-
ance, and to N. El Solh (Centre National de Référence des
Staphylocoques, Institut Pasteur, Paris, France) and W. Witte
Staphylococcus aureus species on the horizon? An update.
Journal of Food Protection (1996) 59:1123–1126
(Robert Koch-Institut, Wernigerode, Germany) for providing 13. Jaulhac B, De Buyser ML, Dilasser F, Prevost G, Piedmont
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Y: Screening of staphylococci for the toxic shock syndrome
toxin-1 (TSST-1) gene. Letters in Applied Microbiology
(1991) 13:90–92
14. Kreiswirth BN, Schlievert PM, Novick RP: Evaluation of
coagulase-negative staphylococci for ability to produce toxic
shock syndrome toxin 1. Journal of Clinical Microbiology
(1987) 25:2028–2029
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