Synthetic exfoliative toxin A and B DNA probes
for detection of toxigenic Staphylococcus
S Rifai, V Barbancon, G Prevost and Y Piemont
J. Clin. Microbiol. 1989, 27(3):504.
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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1989, p. 504-506
Copyright 1989, American Society for Microbiology
Vol. 27, No. 3
Synthetic Exfoliative Toxin A and B DNA Probes for Detection of
Toxigenic Staphylococcus aureus Strains
SAMER RIFAI,* VIVIANE BARBANCON, GILLES PREVOST, AND YVES PIEMONT
Laboratoire de Toxinologie Bactérienne, Institut de Bactériologie de la Faculté de Médecine, Université Louis Pasteur,
3, rue Koeberlé, 67000 Strasbourg, France
Received 28 June 1988/Accepted 6 December 1988
Two methods for the detection of exfoliative toxin (ET) from Staphylococcus aureus were compared: (i) a
phenotypic assay, electrosyneresis, and (ii) a genotypic assay, staphylococcal DNA hybridization with
oligodeoxynucleotide probes. The probes were chosen from the previously determined sequences of serotype A
and B of ET, one probe for serotype A and another for serotype B. Strains exhibiting ET production in
electrosyneresis always possessed the ET gene(s). Conversely, some strains not exhibiting ET production in
electrosyneresis harbored the ET gene(s). The latter strains produced low levels of ET. ET-negative phage
group 2 strains of S. aureus as well as tested coagulase-negative staphylococci did not possess the ET gene(s).
The sensitivity of the DNA hybridization technique was 106 bacteria or 100 ng of genomic DNA.
Staphylococcal exfoliative toxins (ETs) are responsible
for cutaneous injuries referred to as staphylococcal scalded
skin syndrome. These lesions are often encountered in
hospitalized infants as sporadic cases or as small epidemics
(1, 16). Only 5 to 6% randomly isolated Staphylococcus
aureus strains produce such toxins (5, 14). These strains
belong in most cases to phage group 2, but only 40% of phage
group 2 strains synthesize the toxins (4, 5, 16). At the present
time, two different toxin serotypes, A and B (ETA and
ETB), are known. Strains producing ETA only represent
88% of toxigenic S. aureus, followed by strains producing
both ETA and ETB (8%) and then by strains producing ETB
only (4%) (14). For ETA the structural gene is chromosomally
encoded, whereas for ETB it is carried by a plasmid
ET-producing strains are detected by several methods
based mostly on the immunological detection of the toxin in
culture fluid: gel immunoprecipitation (2), enzyme-linked
immunosorbent assay (10), or radioimmunological assay (3,
17). The biological assay, the newborn mouse test, is not
easy to handle, and its sensitivity is only 5 p.g of ET per ml.
Among the immunological methods, electrosyneresis is well
adapted to the epidemiological characterization of ET-producing
S. aureus strains, since this method is rapid and can
be used to test simultaneously many strains (12). However,
strains with low ET excretion could be misidentified, since
the sensitivity of the test is only 5 ,ug/ml. We therefore
compared ET detection by electrosyneresis to ET gene
detection by staphylococcal DNA hybridization with DNA
probes. The latter technique allows the detection of ET
gene-harboring strains, whatever expression they have.
MATERIALS AND METHODS
Staphylococcal strains. The staphylococcal strains tested
included separate sets of wild-type strains previously collected:
98 ETA-producing, 22 ETB-producing, and 29 ETAand
ETB-producing S. aureus strains, 51 ET-negative S.
aureus strains, 32 phage group 2 S. aureus strains, and 138
coagulase-negative staphylococcal strains. All S. aureus
strains were isolated in our laboratory from various pathological
samples. The coagulase-negative staphylococci were
obtained from urine samples with more than 105 staphylococci
per ml or from N. El Solh; the species were as follows:
38 S. epidermidis, 29 S. haemolyticus, 20 S. saprophyticus,
27 S. hominis, and 24 S. warneri. The reference S. aureus
strains were, for ETA, strain 50586 isolated in our laboratory
and used in previous studies (10, 11, 13) and, for ETB, strain
TC142 obtained from J. P. Arbuthnott, Trinity College,
Electrosyneresis. ET production was assessed by electrosyneresis
(or counterimmunoelectrophoresis) as previously
described (12). Briefly, the strains were cultivated in
0.5 ml of TY medium (6) under an air phase containing 15%
carbon dioxide. Culture supernatant (20 pI) and ETA or ETB
rabbit antisera (20 lI) were used for immunoprecipitation by
electrosyneresis in an electric field of 4 V/cm.
ET production in rabbit peritoneal cavities. Previously
sterilized dialysis bags were filled with phosphate-buffered
saline and seeded with staphylococcal strains. They were
inserted into rabbit peritoneal cavities. After 5 days, the fluid
in the bags was centrifuged and the supernatant was tested
for ET content.
Concentration of culture supernatant. The staphylococci
were grown in 25 ml of TY medium as described above. The
supernatant was placed in a Centricon device (Amicon
Corp.) with a cutoff of 10 kilodaltons and centrifuged for
several hours until a sevenfold concentration was achieved.
DNA probes. The DNA probes were chosen on the basis of
the nucleotide sequences of the ETA and ETB genes (7, 9).
The ETA probe was a 56-mer coding for amino acids -21
through -3 from the signal sequence of the ETA molecule;
it had the following nucleotide sequence: 5'CTGTAGG
AAAAAAACCATGC3'. The ETB probe was a 50-mer coding
for amino acids 222 through 238 of the corresponding
protein. It had the following nucleotide sequence: 5'CAC
GGAACGATTTG3'. The probes were 5' labeled with [-y-32P]
ATP by using T4 polynucleotide kinase (Boehringer Mannheim
Biochemicals) as described by Maniatis et al. (8). The
specific activities were 13 x 106 cpm/,ug for the ETA probe
and 43 x 106 cpm/lpg for the ETB probe.
Dot blots. TY medium (200 pI) was aseptically dispensed
into each of the 96 wells of sterile, round-bottomed micro-
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VOL. 27, 1989
dilution plates. The staphylococcal strains were seeded in
each well with sterile toothpicks and grown overnight at
37°C. The plates were centrifuged for 10 min at 3,000 x g,
and the supernatant was discarded. The pellet was suspended
in 25 ,ul of TE buffer (50 mM Tris, S mM EDTA [pH
8.0]) containing 10 U of lysostaphin and 2 mg of lysozyme
per ml for 60 min at 37°C. Samples (4 ,ul) from each well were
spotted onto nitrocellulose filters (BA 85, 0.45-,um pore
diameter; Schleicher & Schuell, Inc.). The nitrocellulose
sheet was placed onto a 0.5 M NaOH-hydrated bed for 12
min; neutralization was performed with 1 M Tris hydrochloride
(pH 7.4) for 3 min, with 1.5 M NaCI-0.5 M Tris
hydrochloride (pH 7.4) for 3 min, and then with 0.3 M NaCI
for 3 min. The air-dried filters were heated at 80°C for 2 h
under vacuum. The membranes were immersed for 2 h at
55°C in lx SSC buffer (lx SSC is 0.15 M NaCI plus 0.0125
M sodium citrate [pH 7.0]) containing 2 mg of proteinase K
per ml and 0.05 % (wt/vol) sodium dodecyl sulfate. The
filters were washed in 3 x SSC buffer and allowed to air dry.
Prehybridization was carried out for 30 min at 65°C in 6.25 x
SSC. buffer containing 0.5% (wt/vol) bovine serum albumin,
0.5% (wt/vol) polyvinylpyrrolidone, and 1% (wt/vol) sodium
dodecyl sulfate. The filters were transferred into the hybridization
solution (6x SSC buffer containing 1% bovine serum
albumin, 1% polyvinylpyrrolidone, and 1 mM EDTA) containing
0.15 pmol of labeled probe per ml. Hybridization was
performed for 30 min at 65°C, followed by two washes (5 min
each) at 65°C with lx SSC buffer containing 1% sodium
dodecyl sulfate. The filters were exposed to Agfa Curix
X-ray film with an intensifying screen at -70°C for 1 or 2
Sensitivity of DNA hybridization. The sensitivity of DNA
hybridization was assessed in two ways. (i) Dilutions of
purified staphylococcal genomic DNAs from reference
strains 50586 and TC142 were spotted onto nitrocellulose
filters and treated as described above. The DNA quantities
tested ranged from 1 ,ug to 10 pg of ETA- and ETBproducing
strains. (ii) After overnight cultivation, strains
50586 and TC142 were aseptically sonicated for 20 s to
dissociate staphylococcal clusters. CFU were determined by
plating dilutions of the cultures on blood agar, or 200-,ul
culture samples were diluted twofold in phosphate-buffered
saline at 4°C and processed as described above for dot blots.
RESULTS AND DISCUSSION
Both probes produced good hybridization signals with
homologous strains, with negligible background reactions
(Fig. 1). ETA-producing strains did not react with the ETB
probe and vice versa. The probes were therefore specific for
the toxin serotype for which they were constructed. Table 1
reveals an excellent correlation between the phenotypic
detection (electrosyneresis) and genotypic detection (hybridization)
of S. aureus ET. Among 200 S. aureus strains
tested, only 2 discrepancies were noted: 2 strains which
reacted with neither ETA nor ETB antisera harbored sequences
complementary to the ETA probe for the first strain
and to the ETB probe for the second.
In an attempt to increase ET production by these two
discordant strains, we placed them in dialysis bags and
inserted the bags into rabbit peritoneal cavities. In both
cases, ET was detected within the dialysis bags by electrosyneresis.
The ET serotype found agreed with the DNA
hybridization results. The peritoneal dialysis bag technique
may increase ET production for two reasons: (i) the staphylococci
grow in a continuously renewed medium, allowing
SYNTHETIC ETA AND ETB DNA PROBES 505
_ _ a
FIG. 1. Spotting of 33 different cultured strains of Staphylococcus
spp. onto nitrocellulose filters. (A) DNA hybridization with the
ETA oligonucleotide probe. (B) DNA hybridization with the ETB
oligonucleotide probe. Rows: 1 to 3, ETA-producing S. aureus
strains hybridizing specifically with the ETA probe; 4 to 6, ETBproducing
S. aureus strains hybridizing specifically with the ETB
probe; 7 to 9, ETA- and ETB-producing S. aureus strains hybridizing
specifically with both probes; 10, coagulase-negative Staphylococcus
strains with no hybridization signal; 11, non-ET-producing
S. aureus strains with no hybridization signal.
the achievement of higher bacterial (and hence toxic) concentrations
than those obtained in classical culture media,
and (ii) physicochemical factors achieved in vivo may be
more suitable for ET gene expression. The role of C02, for
example, was monitored in vitro (10): its concentration in the
air phase was critical for ET production in TY medium.
N N %
Detection of ET-producing staphylococcal strains by
electrosyneresis and hybridization
No. of strains detected
Species and ET Electro- Hybrd Discrepancies
serotypea (no. of syneresis ybri iza- between methods
strains tested) with probe (no. of strains)
A B A B
ETA (98) 98 0 98 0 0
ETB (22) 0 22 0 22 0
ETA + ETB 29 29 29 29 0
Non-ET 0 0 1 1 2
Coagulase-negative 0 0 0 0 0
a As determined by electrosyneresis.
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506 RIFAI ET AL.
To determine whether the in vitro conditions were suitable
for ET production by these two discordant strains, we
concentrated both culture supernatants. ET was detected in
both concentrates by electrosyneresis. A semiquantitative
estimation showed that, with respect to the reference
strains, the ET concentrations were reduced by about 2 log
Strains with weak ET expression in vitro are difficult to
detect by immunoprecipitation, even with culture conditions,
such as a 15% C02 concentration in the air phase,
favoring ET production. These strains, however, can be
responsible for cutaneous injuries, since ET is significantly
produced in vivo. Staphylococcal DNA hybridization with
probes for ET can detect these fastidious strains.
As the bulk of ET-producing S. aureus strains belong to
phage group 2, we wondered whether all phage group 2
strains harbored the ET gene(s), with or without expression.
Among 32 strains tested belonging to this phage group, only
2 harbored the ETA gene. The same strains (and no others)
expressed this gene, as detected by electrosyneresis; no
hybridization occurred with the ETB probe. These results
indicate that phage group 2 strains do not necessarily possess
the ET gene(s).
Finally, as previously noted with electrosyneresis (14),
hybridization never detected the ET gene in the coagulasenegative
Can such probes be used directly for the detection of
ET-producing strains in pathological samples? To answer
this question, we checked the sensitivity of our method. The
lowest detection limit was 106 lysed bacteria spotted onto
nitrocellulose or 100 ng of purified genomic DNA. This
bacterial number is too high to allow a sensitive diagnosis of
toxigenic staphylococcal infections directly from pathological
samples. The DNA hybridization technique is very
efficient and should be used for the precise study of previously
isolated staphylococcal strains and not for a direct
diagnosis from pathological samples. This method allows the
study of several hundred strains at a time.
This work was supported by grant CRE 861018 from the Institut
National de la Santé et de la Recherche Médicale.
We thank N. El Solh, Centre National de Référence des Staphylocoques,
Institut Pasteur, Paris, France, who generously provided
strains of S. hominis and S. warneri.
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