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Journal of Clinical Microbiology, September 1998, p. 2548-2553, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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
Karsten
Becker,*
Ricarda
Roth, and
Georg
Peters
Institute of Medical Microbiology, University
of Münster, 48149 Münster, Germany
Received 18 December 1997/Returned for modification 10 April
1998/Accepted 22 June 1998
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ABSTRACT |
Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for
Staphylococcus aureus exotoxin gene screening as an
alternative to the conventional biological assays, which depend on
detectable amounts of toxins produced. One set of oligonucleotide
primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC,
entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta
and etb) and the toxic shock syndrome toxin 1 gene
(tst). Oligonucleotide primers were used as published
previously, modified or newly developed to meet the requirements of
both good size-distinguishable amplification bands of multiplex PCR and
the temperature limit of the uracil DNA glycosylase system for
carryover protection. Amplification products were visualized by agarose
gel electrophoresis, and specificity was controlled with the aid of a
DNA EIA system using oligonucleotide probes derived from the sequences
of the S. aureus toxin genes. PCR procedures were performed
by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical
strains. The PCR results were compared with those of immunological
toxin production assays. This multiplex PCR-EIA system offers an
alternative method for the rapid, sensitive, specific, and simultaneous
detection of the clinically important exotoxin potency of isolated
S. aureus strains for diagnostic purposes as well as
research studies.
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INTRODUCTION |
Many Staphylococcus
aureus strains produce one or more of a group of specific
exotoxins that include staphylococcal enterotoxins (SEs),
staphylococcal exfoliative toxins (ETs), and toxic shock syndrome toxin
1 (TSST-1). The SEs, which cause staphylococcal food poisoning, are
classified by serological criteria into five major groups (A to E,
referred to as SEA to SEE, respectively), and SEC can be further
subdivided into SEC1, SEC2, and
SEC3, based on differences in minor epitopes (1,
3, 4, 6, 11, 12, 23). The ETs are responsible for the
staphylococcal scalded-skin syndrome, and currently two different toxin
serotypes (A and B, referred to as ETA and ETB, respectively) are known
(16, 27). TSST-1 is the major exotoxin etiologically
involved in staphylococcal toxic shock syndrome, especially in
menstrual cases (5, 25).
Conventional methods for the detection of such toxin-producing S. aureus strains are based on immunological procedures measuring the
toxin in the culture supernatants of suspected S. aureus
strains or in contaminated food extracts or in patient specimens
(26). These methods are always dependent on detectable
amounts of toxins. In recent years, protein sequences have been
established for all these toxins, as well as nucleotide sequences for
the corresponding genes (tst, entA,
entB, entC1 to
entC3, entD, entE,
eta, and etb) (2, 8, 10, 13, 14, 18, 20,
22). DNA-DNA hybridization techniques have been developed that
use gene fragments or oligonucleotide probes that react with either one
or more of the respective toxin genes (24). Recently,
specific oligonucleotide primers for PCR have been described for
analysis of S. aureus strains for the presence of toxin
genes, mainly for scientific reasons (9, 17). However, there
is still need for rapid, specific, and, especially, simultaneous
detection of the genes for production of all of these exotoxins of
isolated S. aureus strains for diagnostic and
epidemiological purposes.
Here we report two multiplex PCR enzyme immunoassays (PCR-EIAs)
one
designed to detect the staphylococcal enterotoxin genes, the other
designed to detect the tst gene and the et genes.
The respective PCR protocols were evaluated by using S. aureus template DNA extracted from a panel of 23 toxin-producing
reference strains and a collection of 50 clinically isolated strains.
The specificity was controlled by the aid of a DNA EIA system using
oligonucleotide probes derived from the nucleotide sequences of the
S. aureus toxin genes. PCR results were compared with those
of immunological toxin production assays.
(This study was presented in part at the 35th Annual Meeting of the
Infectious Diseases Society of America, San Francisco, Calif., 1997.)
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MATERIALS AND METHODS |
Bacterial strains.
The 23 toxin-producing reference strains
(see Tables 3 and 4) were American Type Culture Collection strains,
were part of the strain collection of our institute, or were kindly
supplied by W. Witte (Robert-Koch-Institut, Wernigerode, Germany) and
N. El Solh (Institut Pasteur, Paris, France) and had been previously defined by virtue of their respective toxin production. In addition to
standard PCR controls for contamination events, S. aureus
Cowan 1 and Staphylococcus epidermidis ATCC 20044 served as
negative controls, with both run with each set of PCR and DNA-EIA
reactions. All clinical strains were isolated in our diagnostic
laboratory. A total of 50 isolates from 43 patients were identified
biochemically by the automated ID 32 Staph system (API Systems S. A.,
Montalieu Vercieu, France) and confirmed as S. aureus by
Pastorex Staph-Plus (Sanofi Diagnostics Pasteur, Marnes-la-Coquette,
France). For nucleic acid (NA) isolation, S. aureus strains
were subcultured on blood agar. A single colony was then inoculated
into brain heart infusion broth (BHI, Merck, Darmstadt, Germany) and
incubated overnight with shaking at 37°C.
DNA isolation procedures.
Total NAs were isolated from 0.5 ml of a brain heart infusion broth culture. Cells were pelleted by
centrifugation at 5,000 × g for 20 min (Biofuge 13;
Heraeus, Hanau, Germany), resuspended in 185 µl of TE buffer (20 mM
Tris chloride, 2 mM EDTA [pH 8.0]) with 15 µl of recombinant
lysostaphin (15 mg/ml) from AMBI (Tarrytown, N.Y.), and incubated at
37°C for 0.5 h. NAs were subsequently extracted with a QIAamp
tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's
recommendations. NA samples were eluted with distilled water and
adjusted to a final concentration of 1 µg/ml according to
A260 values.
PCR.
Oligonucleotide primers are listed in Table
1. Primarily, we tested primer sequences
published by Johnson et al. (17). Modifications or newly
designed PCR primers were tested to meet the requirements of a
multiplex PCR to obtain good size-distinguishable amplification bands
as well as the requirements of the uracil DNA glycosylase (UNG) system
with an inferior limit of annealing temperature at 55°C. These new or
modified primers and the biotinylated oligonucleotide probes were
designed by computerized sequence analysis with PC/Gene, release 18.0 (IntelliGenetics, Mountain View, Calif.) and Primer Premier 4.04 (Premier Biosoft, Palo Alto, Calif.). The oligonucleotides were
obtained from MWG Biotech (Ebersberg, Germany).
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TABLE 1.
Base sequences, gene locations, and predicted sizes of
PCR products for the S. aureus exotoxin-specific
oligonucleotide primers
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First, preliminary trials with different annealing temperatures (50, 52.5, 55, and 57.5°C), cycle numbers (25, 30, and 35),
amounts (1.0, 1.25, 1.5, and 2.0 U), and brands of DNA polymerases
(
Taq
DNA polymerase; Boehringer, Mannheim, Germany; Expand high-fidelity
PCR
system, Boehringer; PrimeZyme, Biometra, Göttingen, Germany;
AmpliTaq DNA polymerase, Applied Biosystems, Foster City,
Calif.;
and
AmpliTaq Gold DNA polymerase, Applied
Biosystems) and with
various concentrations of magnesium chloride (1.0, 1.5, 2.0, 2.5,
3.0, 3.5, and 4.0 mM) were performed to define the
optimal PCR
conditions (data not shown).
The amplification was performed in an automated thermocycler with a hot
bonnet (Hybaid, Teddington, United Kingdom). The optimized
thermal
cycling conditions for both multiplex PCRs were 30 cycles
of
denaturation at 95°C for 1 min (2 min for the first cycle),
annealing at 55°C for 1 min, and polymerization at 72°C for 2
min
(5 min for the last cycle). Optimized amplifications were
carried out
in 0.5-ml tubes in a reaction volume of 50 µl containing
200 µM
dATP, dCTP, and dGTP. Instead of dTTP, dUTP was used in
a concentration
of 600 µM. The master mix (PCR Core Kit Plus;
Boehringer) contained
10 mM Tris-HCl (pH 8.9), 50 mM KCl, 3 mM
magnesium chloride, 1.0 U of
UNG, and 0.5 pmol of primer. This
reaction mixture was incubated for 10 min at room temperature
(UNG reaction condition). For UNG inactivation,
prevention of
primer-dimer formation and denaturation of template DNA,
after
addition of template DNA (5 to 10 ng), the mixture was incubated
at 95°C for 5 min and stored on ice before addition of 1.25 U
of
Taq DNA polymerase (Boehringer, Mannheim, Germany).
Amplified products (10 µl) were resolved by 3.0% agarose gel
electrophoresis at 150 V for 1 to 2 h. The gel was then stained
with ethidium bromide, and the bands were visualized under UV
illumination at 254 nm.
Hybridization procedures.
Designed 5'-biotinylated
oligonucleotide probes for the eight toxins tested (Table
2) were separately used for hybridization of amplified DNA from the multiplex PCRs in a generic DNA EIA (GEN-ETI-K DEIA; Sorin, Saluggia, Italy) according to the
manufacturer's recommendations. Briefly, strips coated with
streptavidin were incubated with an optimized concentration of the
biotinylated probes (20 pg/µl for each probe) at 5°C overnight.
After addition of 100 µl of hybridization buffer, the strips were
incubated with 20 µl of the 1:10-diluted denatured PCR products at
the optimal hybridization temperature (55°C for all used probes) for
1 h. Following hybridization, the samples were treated with 100 µl of a 1:50-diluted anti-double-stranded DNA mouse antibody solution for 1 h. The detection of the DNA-antibody bond was performed by
addition of an enzyme tracer system (antimouse antibody labelled with
horseradish peroxidase), and the results were measured after incubation
with the chromogen-substrate solution by using a vertical reading
photometer. The optical density at 450/630 nm (OD450/630 ratio) of the resulting color was measured, and the
A630 was subtracted from the
A450. A cutoff of 0.150 absorbance units above
the mean value of determinations of toxin-negative reference strains
was defined as a positive reaction.
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TABLE 2.
Base sequences and gene locations for the S. aureus exotoxin-specific 5'-biotinylated oligonucleotide probes
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Sensitivity testing.
For determination of the sensitivities
of the PCR assays and hybridization procedures, the minimal amount of
staphylococcal DNA that would be successfully detected was determined.
Purified DNA of reference strains was quantitated by using a
spectrophotometer, and 10-fold serial dilutions starting at 1 µg/ml
were subsequently subjected to multiplex PCR and DNA EIA. Upon
completion of PCR, an aliquot from each tube was analyzed by
electrophoresis and placed into the DNA EIA as described above. To
compare the influence of the multiplex procedure versus that of a
single-PCR application, DNA of the toxin-positive reference strains was
tested as a single target versus equal amounts of a mixture of all gene
targets.
Specificity testing.
Samples of DNA from the target
toxin-negative S. aureus strain Cowan 1 and from a set of
coagulase-negative staphylococci, from pyrogenic exotoxin and
streptolysin O-producing strains of Streptococcus pyogenes,
and from other gram-positive microorganisms (microcooci, stomatococci,
and bacilli) of the strain collection of our institute were tested. Ten
nanograms of template DNA was used per reaction.
Determination of staphylococcal toxins.
Culture filtrates of
the S. aureus strains were tested for the presence of SEA to
SEE by an EIA (Ridascreen set A, B, C, D, E; R-Biopharm, Darmstadt,
Germany), and, except for SEE, by a semiquantitative reversed passive
latex agglutination test (SET-RPLA; Unipath, Basingstoke, Hampshire,
United Kingdom). The presence of TSST-1 was determined by the same
method (TST-RPLA; Unipath). An immunoblot with sheep anti-ETA antibody
(Toxin Technology, Sarasota, Fla.), alkaline phosphatase-conjugated
donkey anti-sheep immunoglobulin G (Sigma, St. Louis, Mo.), and
purified ETA (Toxin Technology) as a positive control was used for the
detection of ETA production.
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RESULTS |
Optimization of PCR conditions.
Titration of Mg2+
concentration of the reaction buffer revealed an optimum of 3 mM for
both multiplex PCRs. The primer sets allowed PCR amplification of the
target gene fragments with 25, 30, or 35 cycles. Thirty cycles was
found to be optimal for both multiplex systems. The optimal
thermal profile was 95°C for 1 min, 55°C for 1 min, and
72°C for 2 min for both multiplex PCRs. The Expand High Fidelity PCR
system (Boehringer, Mannheim, Germany) and the use of other DNA
polymerases had no advantages over our multiplex PCR systems.
DNA EIA.
Analysis of the nucleotide sequences published for
the target genes indicated that the 24- to 26-base oligonucleotide
probes (Table 2) encode sequences unique to the toxins from which they were deduced. Hybridization experiments with these gene probes were
performed with PCR amplification products derived from single and
multiplex PCRs as described above. The optimal concentration for
coating the microtiter plates with the biotinylated probes was 20 pg/µl for each probe. The results indicated a complete correlation
between the amplification products and their corresponding hybridization. No unspecific hybridization was observed among the
different PCR products of all staphylococcal exotoxins tested.
Sensitivity of multiplex PCR and DNA EIA.
Both multiplex
primer sets successfully amplified the appropriate regions of the
target toxin genes from a DNA dilution containing as little as 50 pg
(ranging from 1 to 50 pg) for single PCRs and in multiplex PCRs as
little as 100 pg (ranging from 1 to 100 pg) of DNA of a DNA mixture
isolated from reference strains containing the test toxin genes. In a
given multiplex PCR mixture consisting of DNA from every type of target
gene, the resulting band intensities of the larger amplicons were
reduced compared to the intensities of the smaller amplicons and to the
artificially pooled single PCR, but this does not affect the test in
practice. The sensitivity of subsequent DNA EIAs was always 10 to 100 times higher than that of both the single and multiplex PCRs (data not
shown).
Specificity of the staphylococcal oligonucleotide primers and
probes.
Initially, the specific primer pairs published by
Johnson et al. (17) were tested for their applicability
to multiplex PCR for detection of the staphylococcal toxin genes. To
meet the requirements of good size-distinguishable amplification bands
of multiplex PCR and of the temperature limit of the UNG system for
carryover protection, it was necessary to modify and partly select new
primers directed to specific nucleotide sequences within the target
genes. The eight modified or newly developed pairs of toxin-specific oligonucleotide primers and the eight newly derived toxin-specific biotinylated oligonucleotide probes were selected by computer analysis
with previously described nucleotide sequences for the corresponding
genes of SEA (7), SEB (18), SEC1 to
SEC3 (10, 13), SED (2), SEE
(14), TSST-1 (8), and ETA and ETB (20). A total of 23 reference strains of S. aureus were used as templates for the primer sets, each with 5 to
10 ng of bacterial DNA in the PCR mixture. The DNA of all S. aureus exotoxin-producing reference strains was specifically
amplified by the primers and confirmed by oligonucleotide probes of the
DNA EIA; thus, the corresponding genes were correctly recognized
(Tables 3 and
4). The sizes of the amplicons were
identical to those predicted from the primer design (Table 1). Figures
1 and 2
show representative gels for the PCR product patterns of both multiplex
PCRs. In addition, none of the primer pairs and the corresponding
probes reacted with any strains of non-SE-producing S. aureus strains, other coagulase-positive staphylococci
(S. intermedius or S. schleiferi subsp.
coagulans), a spectrum of coagulase-negative staphylococcal species (S. epidermidis, S. haemolyticus, S. hominis, S. warneri, S. capitis, and S. simulans), and other related gram-positive genera, including
stomatococci, micrococci, and Bacillus spp. (data not
shown). Because of the close relationship between SEB and SEC and other
members of the pyrogenic exotoxin family, we tested pyrogenic exotoxin
and streptolysin O-producing strains of S. pyogenes in
our multiplex PCR system, and no specific amplification and
hybridization were observed.
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TABLE 3.
Results of testing reference strains for staphylococcal
enterotoxin genotype derived from agarose gel analysis of multiplex
PCR and colorimetric microtiter plate DNA EIA
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TABLE 4.
Results of testing reference strains for staphylococcal
ETs and TSST-1 genotype derived from agarose gel analysis of
multiplex PCR-EIA and colorimetric microtiter plate DNA EIA
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FIG. 1.
Agarose gel electrophoresis patterns showing
PCR-amplified products in multiplex PCR for the staphylococcal
enterotoxin genes. Lanes 1, DNA molecular weight marker  HindIII; 2, sea; 3, seb; 4, sec; 5, sed; 6, see; 7, multiplex PCR
with all enterotoxin genes simultaneously (sea to
see); 8, artificial arrangement of the amplification
fragments of sea to see; 9, DNA molecular weight
marker BstE II. Sizes are marked in base pairs on the
left and right.
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FIG. 2.
Agarose gel electrophoresis patterns showing
PCR-amplified products in multiplex PCR for the staphylococcal ET genes
(eta and etb) and TSST-1 gene (tst).
Lanes 1: DNA molecular weight marker HindIII; 2, eta; 3, etb; 4, tst; 5, multiplex PCR
with both ET genes and the TSST-1 gene simultaneously; 8, artificial
arrangement of the amplification fragments of eta,
etb, and tst; 9, DNA molecular weight marker BstE II. Sizes are marked in base pairs on the left and
right.
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Testing of S. aureus isolates by multiplex PCR-EIA
and comparison with toxin phenotype.
Amplicons with predicted
sizes and a positive hybridization reaction were detected in all
reference strains analyzed (Tables 3 and 4). Also, there was 100%
correlation between the results of the multiplex PCR-EIA and the toxin
phenotype testing of the reference strains.
Within 50 clinical
S. aureus isolates obtained from 43 patients, a total of 19 positive amplification results were found.
All
could be confirmed by the respective hybridization procedure.
In 11 clinical strains from different patients, we detected various
genes for
enterotoxins, in 7 strains we detected the TSST-1 gene,
and in 1 strain
we detected the ETA gene (Table
5).
Both multiplex PCR systems could detect exotoxin double producers in
reference strains (one SEA plus SED and one ETA plus
ETB) in one step.
In the clinical strains, we observed five strains
carrying two exotoxin
genes: one strain carried the
sec and
tst genes,
and four strains carried the
sea and
tst genes
(Table
5).
Thus, an excellent correlation
was found between the results of
genotype and phenotype testing. In
only one strain positive for
sea by multiplex PCR-EIA could
SEA toxin production not be detected
by both immunological tests used.
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TABLE 5.
Detection of exotoxin-producing S. aureus
reference strains (n = 23) and clinical isolates
(n = 50) by immunoassays and multiplex PCR-EIA
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DISCUSSION |
The ability of S. aureus strains to produce SEs,
ETs, and/or TSST-1 is an important property with various clinical
implications. Determination is still mainly based on
immunological methods for toxin detection which are time- and
labor-consuming. Furthermore, these methods depend on the
concentration of toxin expressed and thus can be negatively influenced
by various factors. Also, for enterotoxins, differences in the toxin
production levels by S. aureus strains grown in natural
substrate and laboratory media have been described previously
(15).
We have developed a multiplex PCR-EIA test system which allows the
simultaneous detection of the enterotoxin (sea to
see), ET (eta and etb), and TSST-1
(tst) genes. We could prove this system to be sensitive and
specific for the respective genes by using staphylococcal cultures.
Because our objective was to develop a simple and fast method for use
in routine diagnosis, we preferred an NA preparation procedure that was
simpler than the classical phenol-chloroform extraction method. Samples
for PCR can be prepared in 1 h without an obvious risk of sample
cross-contamination. The use of alternative DNA polymerase systems,
so-called expanded and/or high-fidelity systems, had no advantages over
our multiplex PCRs, although they are described as being able to
increase the yield of amplification by reducing the mismatch pausing
associated with Taq DNA polymerase.
Strict precautions must be taken to avoid false-positive amplification
because of contamination of the reaction mixture both with target DNA
from staphylococcal cells and by carryover of amplified target DNA from
previous PCR cycles. Therefore, to prevent false-positive results, we
used the UNG system (21). This enzyme can be used with dUTP
to eliminate PCR contaminations from previous DNA synthesis reactions
by degrading uracil-containing DNA. Since it has been reported that
UNG from Escherichia coli remains partially active or
regains activity leading to degradation of the dU-containing PCR
product, it is necessary to keep the reaction mixture at a higher
temperature (70°C) or to freeze the product immediately after the
last amplification step (19). Because UNG shows
activity only below 55°C, it is recommended to choose annealing
temperatures at or above this temperature in order to avoid degradation
of newly synthesized dU-containing amplicons by possible residual UNG
activity after the inactivation step of this enzyme. To meet this
recommendation, we had to modify or design new primers composed of
nucleotides allowing such temperatures. A higher annealing temperature
is also recommended to increase the specificity of the PCR. The use of
a DNA EIA system as a hybridization step after the PCR procedure offers
a simple and reliable specificity control of the amplicons.
The multiplex PCR-EIA test system described here was primarily designed
for the detection of exotoxin genes in S. aureus in culture. Research is in progress to apply this system to the direct detection of toxigenic S. aureus strains in human
specimens and food material.
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ACKNOWLEDGMENTS |
We thank Susanne Deiwick and Martina Schulte for excellent
technical assistance. We are grateful to M. Herrmann for critical suggestions on the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: University of
Münster, Institute of Medical Microbiology, D-48149
Münster, Germany. Phone: (49) 251 83-55360. Fax: (49) 251 83-55350. E-mail: kbecker{at}uni-muenster.de.
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Journal of Clinical Microbiology, September 1998, p. 2548-2553, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
