Simultaneous Analysis of Multiple Staphylococcal Enterotoxin Genes by an Oligonucleotide Microarray Assay

Abstract

Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.

Staphylococcal food-borne diseases resulting from the consumption of food contaminated with staphylococcal enterotoxins (SEs) are one of the most common food-borne illnesses (1, 5, 11, 14, 31). SEs are also involved in rheumatoid arthritis (17, 38), atopic eczema (9, 10, 27), and toxic shock syndrome (16). SEs are considered potential bioweapons.

SEs belongs to a protein family called superantigens, which induce a polyclonal immune response by direct binding to class II major histocompatibility complex proteins and T-cell receptors on the surfaces of B and T cells without being internalized and processed like a normal antigen (3, 15, 28). These toxins may be involved in modulating the host immune response and may contribute to evasion of host defenses and bacterial persistence (12). Expression of specific enterotoxin (ent) genes by Staphylococcus aureus depends on the host tissue source and may play a role in the adaptation of S. aureus to the host environment (4).

There are 17 known major types of SEs (SEA to SER, respectively, with no SEF), and multiple SEs are commonly found among S. aureus strains (19, 20, 32). Many of the known staphylococcal enterotoxins (SEK to SER) were discovered recently.

The traditional method of identifying SEs by serological typing is relatively complex and time-consuming and is impractical for the detection and identification of a large group of related toxins with significant antigenic similarities (23, 24). Furthermore, the concentrations of toxins produced by S. aureus strains differ when the strains are grown on various natural substrates and laboratory media (7, 33). Other techniques have been used to identify toxin genotypes, including DNA-DNA hybridization and PCR, but these protocols were designed to detect only one or a few toxin genes (21, 35). Multiplex PCR for detection of several ent genes has been reported (6, 26, 29, 30, 36), but additional restriction endonuclease assays or other steps are required to ensure unambiguous identification of ent-specific amplicons. Therefore, there is still a need for a rapid and specific method for simultaneous detection and identification of SEs for diagnostic and epidemiological purposes.

Here we describe a rapid and reliable one-tube microarray-based assay for simultaneous detection and identification (genetic typing) of almost all known ent genes. The method includes PCR amplification of part of the ent genes with universal primers, followed by analysis of amplicons by hybridization with ent-specific oligonucleotide probes immobilized on the microchip.

MATERIALS AND METHODS

Bacterial strains.

The strains used in this study were obtained from the S. aureus collection of the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA; Focus Technologies, Inc., Herndon, Va.) and from the bacterial collection of Farukh Khambaty, Center of Food Safety and Applied Nutrition, Food and Drug Administration (FDA).

Total DNA preparation.

DNA was extracted from freshly grown cells by phenol-chloroform extraction (34). The presence, concentration, and purity of genomic DNA in the prepared samples were detected by measuring the absorbances at 260 and 280 nm with an Ultraspec 3000 spectrophotometer (Pharmacia, Peapack, N.J.).

PCR amplification.

Table 1 lists the primers used to amplify different S. aureus enterotoxin genes. Specific primers were used for amplification of individual enterotoxin genes. The standard PCR mixture (30 μl) contained 1.5 U of HotStar Taq DNA polymerase, 1× buffer supplemented with 2.0 mM MgCl₂ (Qiagen, Valencia, Calif.), 200 nM each forward and reverse primers, 200 μM each deoxynucleoside triphosphate (dNTP; dATP, dGTP, dCTP, and dTTP), and 100 to 300 ng of DNA template. PCR was performed with a Gene AMP PCR system 9600 thermocycler (Applied Biosystems, Foster City, Calif.) with the following cycle conditions: initial activation at 95°C for 15 min; 40 cycles at 94°C for 30 s, 55°C for 40 s, and 72°C for 60 s; and a final extension at 72°C for 7 min. The presence of amplified PCR products was detected by 2% agarose gel electrophoresis in 1× Tris-acetate-EDTA or Tris-borate-EDTA buffer. The gels were stained with ethidium bromide and photographed under UV light with a digital camera (EDAS 290; Kodak, Rochester, N.Y.).

TABLE 1.

Primers used for amplification of S. aureus ent genes

Gene	GenBank accession no(s).	Primers	Nucleotide sequence(5′→3′ end)	Tm (°C)a	Length (nt)b
sea	M18970	setA-F	GGATATTGTTGATAAATATAAAGGGAAAAAAG	51	439
		setA-R	GTTAATCGTTTTATTATCTCTATATATTCTTAATAGT	54
seb	M11118	setB-F	AGATTTAGCTGATAAATACAAAGATAAATACG	54	494
		setB-R	TCGTAAGATAAACTTCAATCTTCACATCT	54
sec	M28364, X05815, AB084256	setC-F	AGATTTAGCAAAGAAGTACAAAGATG	52	490
		setC-R	AAGGTGGACTTCTATCTTCACACTT	54
sed	M28521	setD-F	AGATTTAGCAAAGAAGTACAAAGATG	55	481
		setD-R	CTACTTTTCATATAAATAGATGTCAATATG	52
see	M21319	setE-F	AGATTTAGCAAAGAAGTACAAAGATG	54	473
		setE-R	TGTATAAATACAAATCAATATGGAGGTTCTCT	55
seg	AF064773, AB016487	setG-F	AGAATTAGCTAACAATTATAAAGATAAAAAAG	52	496
		setG-R	TCAGTGAGTATTAAGAAATACTTCCAT	52
seh	U11702	setH-F	TGATTTAGCTCAGAAGTTTAAAAATAAAAATG	52	466
		setH-R	TTTCTTAGTATATAGATTTACATCAATATG	51
sei	AF064774	setI-F	TGATTTAGCTCAGAAGTTTAAAAATAAAAATG	52	505
		setI-R	TTAGTTACTATCTACATATGATATTTCGA	52
sej	AF053140	setJ-F	ATGAAAAAAACAATATTTATACTGATTTTCTCCC	55	807
		setJ-R	TCTACAGAACCAAAGGTAGACTTATTAATAC	57
sek	U93688, AF410775, AP004828	setK-F	ATGAATCTTATGATTTAATTTCAGAATCAA	51	545
		setK-R	ATTTATATCGTTTCTTTATAAGAAATATCG	51
sel	AF217235	setL-F	ATGAAAAAAAGATTATTATTTGTAATTGTTATTAC	52	723
		setL-R	ATCATCTTTTTGAAATTTCGACATCTAG	53
sem	AF285760, AP003363	setM-F	ATGAAAAGAATACTTATCATTGTTGTTTTATTG	53	720
		setM-R	CTTCAACTTTCGTCCTTATAAGATATTTC	54
sen	AF285760	setN-F	ATAAAAAATATTAAAAAGCTTATGAGATTGTTC	52	777
		setN-R	ACTTAATCTTTATATAAAAATACATCAATATG	50
seo	AF285760	setO-F	TATGTAGTGTAAACAATGCATATGCA	53	685
		setO-R	TCTATTGTTTTATTATCATTATAAATTTGCAAAT	53
sep	AP003135	setP-F	TTAGACAAACCTATTATCATAATGGAAGT	52	618
		setP-R	TATATAAATATATATCAATATGCATATTTTTAGACT	53
seq	U93688, AF410775	setQ-F	GGAAAATACACTTTATATTCACAGTTTCA	53	539
		setQ-R	ATTTATTCAGTTTTCTCATATGAAATCTC	52
		Uni1 P1F	TRYAYRTAYGGIGGDVTHAC	40-53	323-377
		Uni2 P2R	AHHRTTYTATTRTCWYYRTAHA	36-51

^aThe basic melting temperature (T_m) was calculated with the Oligonucleotide Properties calculator (http://www.basic.nwu.edu/biotools/oligocalc.html).

^bAmplicon size (in nucleotides [nt]).

For simultaneous amplification of multiple enterotoxin genes by PCR with universal primers, the standard PCR mixture (50 μl) contained 5 U of HotStart Taq DNA polymerase (Qiagen), 1× buffer supplemented with 3.5 mM MgCl₂ (Qiagen), the universal primer mixture (700 nM forward universal primer, 1,400 nM reverse universal primer, and the seh-specific reverse primer at a concentration of 50 nM), 200 μM each dNTP, and 600 to 800 ng of template (total DNA). PCR was performed with a Gene AMP PCR system 9600 thermocycler (Applied Biosystems) with the following conditions: initial activation of the enzyme at 95°C for 15 min; 7 cycles at 94°C for 1 min, 40°C for 1 min, and 72°C for 1 min; 35 cycles at 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 7 min. The PCR products were purified with a Qiaquick PCR purification kit (Qiagen). The concentrations of the PCR products were estimated by measuring the absorbance at 260 nm.

Synthesis of ssDNA.

Single-stranded DNA (ssDNA) samples were synthesized by use of a primer extension (PE) reaction in the presence of only the reverse primer. The standard mixture (50 μl) for PE with enterotoxin gene-specific primers contained 3 U of Taq DNA polymerase (Sigma, St. Louis, Mo.), 1× PCR buffer, 200 nM the corresponding reverse primer, 200 μM each dNTP, and 300 to 500 ng of the amplicon obtained during the previous PCR step. PE reactions were performed with a Gene AMP PCR system 9600 thermocycler (Applied Biosystems) with the following temperature conditions: initial denaturing of DNA at 94°C for 2 min, followed by 40 cycles each of 94°C for 30 s, 52°C for 40 s, and 72°C for 1 min and a final extension at 72°C for 7 min.

The PE reaction mixture for multiple ent genes with a universal reverse primer (700 nM) and seh reverse primer (50 nM) was the same as described above, except that the amount of DNA template (PCR amplicons) was increased to 800 ng to 1 μg. The cycling conditions were the following: initial activation of the enzyme at 95°C for 15 min; 40 cycles at 94°C for 30 s, 45°C for 40 s, and 72°C for 60 s; and a final extension at 72°C for 7 min. The ssDNA was purified with a Qiaquick PCR purification kit (Qiagen) and dried under vacuum.

Chemical labeling of ssDNA.

The dry ssDNA was reconstituted in 20 μl of water and chemically labeled with a fluorescent dye (cyanine 5 [Cy5]) with a MicroMax labeling kit (Perkin-Elmer, Boston, Mass.), according to the protocol of the manufacturer. Nonincorporated dye was removed from the DNA by purification through Centrisep columns (Princeton Separations, Adelphia, N.J.). The amount of the Cy5 dye incorporated into ssDNA was monitored by measuring the ratio of the absorbance at 649 to the absorbance at 260 nm. The typical ratio of λ₆₄₉/λ₂₆₀ was about 0.15 to 0.25, which corresponds to 1.5 to 3 dye moieties per 100 nucleotides of ssDNA.

Design of PCR primers and enterotoxin gene-specific microarray oligonucleotide probes.

Searches with the BLAST program were used to find and retrieve the sequences of the available ent genes. The retrieved sequences were aligned by using ClustalX software (37). Sequences of highly conserved regions among all alleles of each ent gene were selected to design toxin-specific primers for accurate detection and identification of each target toxin gene. Toxin-specific oligonucleotide probes were designed by using highly conserved regions for alleles of each ent gene within the region flanked by primers. The oligonucleotides selected are summarized in Table 2. The 5′ end of the amino acid sequence of each oligonucleotide probe was modified during the synthesis (Qiagen) to enable the immobilization of the oligonucleotide to silylated (aldehyde) slides (ArrayIt, Sunnyvale, Calif.).

TABLE 2.

Oligonucleotide probes for detection and discrimination among ent genes

Probe	Sequence	Length (nt)a	Tmb (°C)
A-1	CGATCAATTTATGGCTAGACG	21	50
A-2	ACTCTGATGTTTTTGATGGGAAG	23	52
A-3	GTTTCATACTTCTACAGAACCTTC	24	52
A-4	ATTCAAATACACTATTAAGAATATATAGAG	30	51
B-1	AATATAGAAGTATTACTGTTCGGGT	25	51
B-2	AGAAGTATTACTGTTCGGGTATTT	24	51
B-3	AAACTCTATGAATTTAACAACTCG	24	49
B-4	ATGAGAATAGCTTTTGGTATGACA	24	51
C-1	AACCACTTTGATAATGGGAACTTA	24	51
C-2	TGTACTTATAAGAGTTTATGAAAATAAAAG	30	51
C-3	AGTTTAACAGTTCACCATATGAAACA	26	52
C-4	ATTGAAAATAACGGCAATACTTTTTG	26	50
D-1	TCACTCCACACGAAGGTAATA	21	50
D-2	TCAATTTGTGGATAAATGGTGTAC	24	51
D-3	ATTTAAAATTGTATAATAATGATACTCTCG	30	51
D-4	TTCTGATGGGTCTAAAGTCTCT	22	51
E-1	ACGGAAAATTTGGTTTATATAACTC	25	49
E-2	TGGTTTATATAACTCAGACAGCTTT	25	51
E-3	TAAGGTGCAAAGAGGCTTGATT	23	52
E-4	ATTCTTCTGAAGGGTCCACG	20	52
G-1	TTAATAGTTCAGAAAATGAAAGAGAT	26	49
G-2	AACAATCGACAATAGACAATCACT	24	51
G-3	ATCACTTGGATTTACAATAACTAC	24	49
G-4	ATGGTTCTGCATTTGAATCTGG	22	51
H-1	AAAGGGTGATTGGTGCTAATGT	22	51
H-2	ATGTTTGGGTAGATGGTATTCAAA	24	51
H-3	ATAAAGACAGCGAAATAAGTAAAG	24	49
H-4	AACTCCTAGAGATTACTCATTC	22	49
I-1	TTATCAGGACAATACTTAAATTCTG	25	49
I-2	AGGCAAAGAATATGGATATAAATCT	25	49
I-3	ATTCAGGTTTTAATAATGGGAAAGT	25	49
I-4	GCCTGTAAGTTTTTTGAAAATTTATGAA	28	51
J-1	CTGCATGAAAACAATCAACTTTAT	24	49
J-2	ATAGCATCAGAACTGTTGTTC	21	49
J-3	TATACAACCCTAGTACCTTTGATG	24	52
J-4	ACCTCAAAAGAACCGTTGGTTA	22	51
K-1	GCTACTAACGAATATCTAGATAAAT	25	49
K-2	CATAACGGCACTAAAAAAGGAG	22	51
K-3	ATAATGACACTTTTTCATATGATTTATTCT	30	51
K-4	TATTCTACACAGGAGATGATGG	22	51
L-1	ACAATAAATTAGATTCGCCAAGAAT	25	49
L-2	GCAAGCATCAAACAGTTACAAC	22	51
L-3	ACGGCACTTCTTCAAAATTTTATAG	22	51
L-4	ATGAATGATGGTTCTAATTTCTCG	24	51
M-1	TTAGCAGGTGATTATTTAGAGAAAT	25	49
M-2	TATGGCTTTAATGATACAAATAAAG	25	48
M-3	ATGATGGTTCATCATTTTCTTATG	24	49
M-4	AACAGGACAAGCTGAAAGTTTC	22	51
N-1	AGATGAAGAGAAAGTTATAGGC	22	49
N-2	ATGGTGTCCAACAAGAAGGTT	21	50
N-3	ATACAATAAAGATACCGGTAACAT	24	49
N-4	CTTTCATTCTCATAATCATCAAGAT	25	49
O-1	ATACATTTATCGTTACTACAGATAA	25	48
O-2	ATGACAGAATGACTAGTGATGTA	23	50
O-3	ACTAGTGATGTACAAAAAGGTTAT	24	49
O-4	AGGAAATTTACCAGATCAATATTTG	25	49
P-1	AGACCTTCAGTCAAGACATTATT	23	50
P-2	ACACAGATGCATTTAATGGAAAAATA	26	50
P-3	ATTGAGTTTCACCCTTCTTCTG	22	51
P-4	ATCCAGATACACAGTTGAGGA	21	50
Q-1	TTGGCGAATCAAAATTTAGATAAAC	25	49
Q-2	ACTATATCTACAGACAAAGTTTCT	24	49
Q-3	GGATATCAGTCAAAATTTAATTCTG	25	49
Q-4	TACATACGATTTGTTTTACACCG	23	50
QCprb	TGGCAGAAGCTATGAAACGATATGGG	27	58
Cy3-QC	CCCATATCGTTTCATAGCTTCTGCCA	26	58

^aAmplicon size (in nucleotides [nt]).

^bThe basic melting temperature (T_m) was calculated with the Oligonucleotide Properties Calculator (http://www.basic.nwu.edu/biotools/oligocalc.html). The references for the oligonucleotide probe are cited in Materials and Methods.

Microchip design and fabrication.

To increase the confidence in the results of the microarray analysis, four individual oligonucleotide probes were selected for each ent gene. To facilitate interpretation of the microarray data, all oligonucleotide probes specific for one gene were placed on a separate row of the array.

Microchips were printed by use of a contact microspotting robotic system (PIXSYS 5500; Cartesian Technologies, Inc., Irvine, Calif.). The average size of the spots was 250 μm. The concentrations of the oligonucleotide probes were adjusted to 100 μM in 50% dimethyl sulfoxide before they were printed on the slides. A quality control oligonucleotide probe (39) of nonbacterial origin was added to each oligonucleotide probe at a concentration of 10 μM to enable monitoring of the spotting and hybridization steps of the microarray assay. Printed slides were incubated for at least 10 min at 85°C to evaporate the dimethyl sulfoxide completely, followed by 15 min of incubation in a freshly prepared 0.25% NaBH₄ solution in water. The slides were washed once for 5 min with 0.1% sodium dodecyl sulfate in water and five times for 1 min each time with distilled water to remove unbound oligonucleotides. Control spots used to mark the array position on the slide were generated by using 1× Spotting Solution (ArrayIt) in 0.25 M acetic acid.

Hybridization conditions.

Hybridization of the fluorescently labeled DNA samples to the microarray was performed in 1× hybridization buffer (5× Denhardt's solution, 6× SSC buffer [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% Tween 20) at 45°C for 45 min. Before hybridization, 2 to 3 μl of Cy5-labeled DNA sample was mixed with an equal volume of 2× hybridization buffer containing 0.1 μM Cy3 quality control probe, followed by denaturation at 95°C for 3 min and chilling on ice. Each sample was placed on the microchip and covered with a glass coverslip (6 by 15 mm) to prevent evaporation of the probe during incubation. After the hybridization, the coverslips were washed away with 6× SSC containing 0.2% Tween 20 at room temperature. The slides were washed in a stepwise manner with 6× SSC buffer, 2× SSC buffer, and 1× SSC buffer for 2 min each and dried by airflow.

Microarray scanning.

Fluorescent images of the microarrays were taken by scanning the slides with a ScanArray 5000 instrument (Perkin-Elmer). The fluorescent signals from each spot were measured and compared by using QuantArray software (Perkin-Elmer).

Sequencing.

We sequenced some enterotoxin genes, including seb, sed, see, and seq. The PCR-amplified DNA fragments were purified by agarose gel electrophoresis, extracted with a QIAquick gel extraction kit (Qiagen) according to the protocol of the manufacturer, and sequenced with an ABI Prism 310 Genetic Analyzer System (PE Applied Biosystems, Foster City, Calif.).

Nucleotide sequence accession numbers.

The accession numbers of the sequences deposited in GenBank are AY518386 for seb of strain ATCC 14458, AY518387 for sed of strain NTCC10656, AY518772 for sem of strain ATCC 19095, and AY518388 and AY518389 for see and seq of strain ATCC 27664, respectively.

RESULTS

Enterotoxin gene-specific PCR primers and microarray oligonucleotide probe designs.

For our genotyping scheme for simultaneous detection and identification (genetic typing) of multiple SE (ent) genes, we developed PCR amplification assays for 16 of the 17 known enterotoxin genes (sea to see and seg to seq) and an oligonucleotide microarray for the identification of the PCR amplicons.

To develop toxin-specific PCR primers and microarray oligonucleotide probes, we performed multiple-sequence alignment analysis of the ent genes using sequence data from GenBank. As shown in Fig. 1, the analysis identified conserved regions flanking variable regions. The conserved regions were used to design universal primers for simultaneous amplification of multiple ent genes. The genetically divergent regions were used to design individual PCR primers specific for each ent gene and to design gene-specific oligonucleotide probes to discriminate among the 16 ent genes (Fig. 1).

FIG. 1.

Multiple-sequence alignment analysis of ent genes. The DNA sequences of 16 major SEs were retrieved from GenBank and aligned by using ClustalX software. The alignment results were presented by using GeneDoc software. Relatively conserved regions that were used for universal primer design are marked with arrows. The sequences of the oligonucleotide probes used for discrimination of the ent genes were selected from within the variable region flanked by the conserved regions. Gray background indicates similar sequences, and black background indicates conserved sequences.

Our sequence data analysis revealed discrepancies in the nomenclatures of several ent genes from the published sequences of strains MW2 and MU50. For example, the sequence named seg in strain MW2 has 98% similarity to seq (GenBank accession number AF410775) and a lower degree of homology to another sequence of seg (GenBank accession number AF064773). Similarly, sep from strain MU50 has only 83% similarity to a different reported sep gene (GenBank accession number NC002745), whereas it has 98% similarity to the sea enterotoxin gene (GenBank accession number M18970).

For the amplification of each ent gene, gene-specific primers were selected on the basis of unique sequences common to all alleles of each toxin gene determined by the multiple-sequence alignment (Fig. 1). To minimize cross-amplification between different toxin genes, the primers selected contained five and more mismatches with homologous toxins. However, in the case of s, we decided not to develop allele-specific oligonucleotide probes for the three described alleles, s, sec1, and sec2, because of their sequence similarities. In the assay, all three are treated as a single gene, the s gene.

To design the oligonucleotide probes for the microarray, toxin-specific sequences were selected from the variable region identified by the multiple-sequence alignment (Fig. 1). Four individual oligonucleotide probes (21 to 30 nucleotides in length, with an average melting temperature of 50°C) were designed to represent the sequence of each target ent gene (Table 2). To minimize cross-hybridization with other ent genes, oligonucleotide probes whose sequences had at least three mismatches with the sequence of the genetically closest ent genes were selected.

Microarray analysis of ent genes in reference strains.

For validation of the selected gene-specific primers and oligonucleotide probes, we used three well-characterized S. aureus sequencing strains, N315, MU50 (22), and MW2 (2), which contain most of the known staphylococcal toxin genes (sea, s, seg, seh, sei, sek, sel, sem, sen, seo, and sep). For the four toxins not coded for by these strains, we used three additional reference strains: ATCC 14458 for seb, NTCC10656 for sed and sej, and ATCC 27664 for see and seq.

Genomic DNA from the five reference strains (N315, MW2, ATCC 14458, NTCC10656, and ATCC 27664) was amplified with the ent-specific primers. The sizes of the PCR products generated with ent-specific primers varied from 466 to 807 bp, depending on the ent gene (Fig. 2A to C), and there was good agreement between the observed and the predicted sizes of the amplicons. We unambiguously identified the presence of all 16 toxin genes previously shown in these strains: the toxin genes s, seg, sei, sel, sem, sen, seo, and sep were found in strain N315 (Fig. 2A); the toxin genes sea, s, seh, sek, sel, and seq genes were found in strain MW2 (Fig. 2B); and the toxin gene seb was amplified from ATCC 14458, the toxin genes sed and sej were amplified from NTCC10656, and the toxin gene see was amplified from ATCC 27664 (Fig. 2C). The toxin genes sea, s, seg, sei, sel, sem, sen, and seo were found in strain MU50 (data not shown). We confirmed the identities of the seb, sed, see, and seq amplicons by sequencing using the corresponding toxin-specific primers. The GenBank accession numbers of the deposited sequences are presented above, in Materials and Methods.

FIG. 2.

PCR amplification of staphylococcal ent genes with gene-specific primers. Total DNA from reference strains N315, MW2, ATCC 14458, NTCC10656, and ATCC 27664 was amplified with gene-specific primers, and the resulting products were separated by electrophoresis on a 2% agarose gel. Lanes: Mr, molecular size markers (indicated on the left in nucleotides); A, sea; B, seb; C, s; D, sed; E, see; G, seg; H, seh; I, sei; J, sej; K, sek; L, sel; M, sem; N, sen; O, seo; P, sep; Q, seq. (A) Strain N315 DNA amplification with all 16 gene-specific primers; (B) strain MW2 DNA amplification with all 16 gene-specific primers; (C) amplification of seb for strain ATCC 14458, sed and sej for NTCC10656, and see for ATCC 27664.

The ent microarray was prepared by immobilizing the four oligonucleotides specific to each of the 16 ent genes in separate rows (shown schematically in Fig. 3I). The left-hand rows contain probes specific for sea to sei, and the right-hand rows contain probes specific for sej to seq. The quality control scan of the array (Fig. 3II) shows the actual scan of such an array. In our quality control procedure (40), each spot was printed with a quality control oligonucleotide, in addition to the specific oligonucleotide probe. Each Cy5-labeled target ssDNA was spiked with quality control Cy3-labeled reference material complementary to the quality control oligonucleotide. The Cy3 quality control scan provides a control image of the entire microarray, which allows validation of the fabrication and hybridization steps.

FIG. 3.

Layout of staphylococcal ent genes on the microarray. Each microarray is composed of four sections, and each section contains four different oligonucleotide probes specific for each of four different ent genes in the section. (I) Schematic illustration of the ent gene microarray. Genes sea to sei are in the rows on the left, and genes sej to seq are in the rows on the right, with each gene represented by four spots (columns). The first columns of each array are printed with spotting solution (U) to assist with array orientation. (II) Quality control scan of an array. Each spot of the array is visualized because each spot contains a small amount of quality control oligonucleotide probe QCprb, which hybridizes with a Cy3-labeled oligonucleotide, Cy3-QC, which is used to spike each target mixture. The spot is visualized with a 543-nm laser. (III) Schematic showing microarray detection of sep. (IV) Microarray analysis of sep genomic DNA from strain N315, which was amplified with sep-specific primers. The resulting PCR product was fluorescently labeled with Cy5 and hybridized to the ent gene array, which was then scanned with a 632-nm laser.

The chip contains 10 identical microarrays for simultaneous analysis of five different microbial samples, each with two duplicates (data not shown).

For microarray analysis of the ent-specific primers amplicons, DNAs from S. aureus reference strains (strains N315, MU50, MW2 ATCC 14458, NTCC10656, and ATCC 27664) were amplified by using the ent gene-specific primers, and fluorescently labeled ssDNA was synthesized from each of the ent amplicons by PE of the PCR products (see Materials and Methods).

The microarray accurately detected each toxin gene. For example, the detection of sep is shown schematically in Fig. 3III, and the actual results from microarray hybridization are shown in Fig. 3IV. It is noteworthy that occasional cross-reacting spots are observed, such as a sel spot (Fig. 3IV, spot L2), which cross-reacted with sep amplicons because of sequence similarity. However, because four oligonucleotide probes were used to detect each ent gene, a small number of cross-reacting spots did not interfere with toxin identification. Figure 4 shows the results of microarray analysis of all 16 ent genes (sea to seq) amplified by specific primers.

FIG. 4.

Microarray-based detection of 16 staphylococcal ent genes with gene-specific primers. Total DNAs from five S. aureus reference strains (strains N315, MW2, ATCC 14458, NTCC10656, and ATCC 27664) were amplified with the ent gene-specific primers, with the panel labels A to E and G to Q corresponding to the lanes described in the legend to Fig. 2. The ssDNAs derived from the PCR products were labeled with Cy5 and hybridized to the ent gene microarray (Fig. 3), which was then scanned with a 632-nm laser.

Overall, the results demonstrate the ability of the microarray to amplify each ent gene with the gene-specific primers and to unambiguously discriminate among the ent genes.

Development of one-tube assay for typing ent genes.

Our attempts to combine all 16 primer pairs in one tube for a multiplex PCR assay did not succeed in amplifying all target ent genes. Several amplicons were not represented; and the yields of seo, sen, seh, and sej were too low to be detected by microarray hybridization (data not shown). This is likely due to interference between primers that significantly reduces the levels of amplification of particular toxin genes (data not shown).

We overcame this problem by developing degenerate primers (universal primers) corresponding to the highly conserved regions of ent (Table 1) and by adding a primer specific for the underrepresented seh gene to the universal primer set. This combination significantly improved the representation of all 16 ent genes. Control DNA and the five individual strains used in the mixture are presented in three-by-two matrix composite image (Fig. 5). All 16 ent genes in the mixture were detected in this analysis (Fig. 5AI). Interestingly, ATCC 14458 (Fig. 5BIII), which is known to code for seb, was found to contain the recently discovered sek and seq genes. Similarly, NTCC10656 was found to encode the seg, sei, sej, sem, sen, and seo genes, in addition to the sed gene, the presence of which in NTCC10656 was already known (Fig. 5BI); and the seq gene was unexpectedly found in ATCC 27664 (Fig. 5BII).

FIG. 5.

One-tube microarray-based detection of 16 staphylococcal ent genes with universal primers. Genomic DNAs from five S. aureus reference strains (strains N315, MW2 ATCC 14458, NTCC10656, and ATCC 27664) were amplified with a mixture of ent gene-specific universal primers supplemented with the primers specific for seh. The resulting PCR amplicons were subjected to PE with a mixture of the reverse ent gene-specific universal primers supplemented with the reverse primers specific for seh, followed by Cy5 chemical labeling. The labeled targets were hybridized to the ent gene microarray, which was then scanned with a 632-nm laser. A control array which contains a mixture of DNA representing all genes (from strains N315, MW2 ATCC 14458, NTCC10656, and ATCC 27664) was included for demonstration of the ability of the assay to detect 16 ent genes in a single sample. AI, strain control array; AII, strain N315; AIII, MW2; BI, NTCC10656; BII, ATCC 27664; BIII, ATCC 14458.

Presence of newly discovered ent genes in staphylococcal isolates analyzed previously.

As shown above, our microarray assay detects multiple ent genes simultaneously and detects additional ent genes in isolates analyzed previously. We used the microarray to analyze additional S. aureus isolates analyzed previously and found that other isolates contained multiple genes in several different combinations (Fig. 6). Strain FRI109, listed in the NARSA collection as coding for sec2, also contains sed, seg, sei, sej, sel, sem, sen, and seo (Fig. 6AII). Strain A900322, which was thought to contain only the enterotoxin gene cluster with seg, sei, sen, seo, and sem (20), also contains the sep gene (Fig. 6BI). Strain MNDON, which was reported in the NARSA catalogue to code for sec1, seg, seh, and sei, is shown here to also encode sel, sem, sen, and seo (Fig. 6BII).

FIG. 6.

One-tube microarray-based analysis of previously analyzed S. aureus strains. Genomic DNA from four strains analyzed previously (strains Mu50, FRI361, A90322, and MNDON) was amplified by using a mixture of ent gene-specific universal primers supplemented with the primer specific for seh. The resulting PCR amplicons were subjected to PE with a mixture of the reverse ent gene-specific universal primers supplemented with the reverse primer specific for seh, followed by Cy5 chemical labeling, and were then hybridized to the ent gene microarray. The resulting image was scanned with a 632-nm laser. The analysis of the four strains is presented in a two-by-two matrix composite image. AI, strain Mu50; AII, strain FRI361; BI, A90322; BII, MNDON.

DISCUSSION

The traditional method of identifying SEs is serological typing with antibodies. In general, serological typing is less sensitive to small variations among SEs than DNA-based methods. Several PCR-based methods are available for S. aureus toxin typing (21, 35). Most require several separate reactions to distinguish among several ent genes. More recently, methods for S. aureus toxin typing by multiplex PCR have been reported (6, 26, 29). These PCR methods are based on combinations of ent gene-specific primers or a combination of universal forward primers and specific reverse primers (36).

One problem with all present PCR-based methods is that novel or unexpected toxin genes can lead to false-positive or -negative results. For example, we observed that all sea gene-specific primers described in the literature can be used for successful amplification of sep as well. This might lead to the mistaken conclusion that a strain encodes sea and incorrect data about the distributions of ent genes and their roles in food poisoning. Since the relationship between the presence of a specific enterotoxin (or a combination of enterotoxins) and human food poisoning is not clearly understood, there is a need for a reliable and universal method for unambiguous identification of known ent genes and for detection of novel ent genes.

We describe here a combination PCR-microarray assay for detection and identification of ent genes. The analysis is based on PCR amplification of a variable region of almost all known ent genes with a single set of degenerate primers whose sequences correspond to those of the flanking highly conserved regions. The amplicons are then identified by analysis on the oligonucleotide microarray. This combined method takes advantage of the strengths of each technique. PCR amplification is highly sensitive, detecting target genes from genomic DNA even when they are present at low concentrations. DNA-DNA hybridization on the microarray increases the specificity of the assay and allows parallel analysis of multiple sequences simultaneously. In addition, the nonspecific amplicons often seen in PCRs have no effect on the hybridization of the targets with specific oligonucleotide probes.

Microarrays are not in common use in average laboratories today. However, like any new technology, as more applications are developed for the microarray technology, it will become more practical and may well become widely used. In the work described here, the presence of genes for each of 16 ent genes was analyzed by four methods: PCR amplification with primers specific for each of the 16 enterotoxin genes, followed by analysis by gel electrophoresis (Fig. 2); PCR amplification with specific primers (Fig. 4), followed by analysis by the microarray assay; amplification of the 16 genes with universal primers, followed by microarray analysis (Fig. 5); and sequencing of the enterotoxin genes to verify their identities. Thus, using two amplification methods (methods with specific primers and universal primers) as well as three DNA analysis methods (gel electrophoresis, DNA sequencing, and DNA microarray analysis), we verified the performance of the method. Using this array, we have shown that some strains previously analyzed by immunological methods contain additional ent genes not detected by the original assays (Fig. 5 and 6).

In our microarray system, we used relatively short oligonucleotides (21 to 30 nucleotides) for three reasons. First, shorter oligonucleotide probe sequences (<25 bp) are often capable of detecting a single-nucleotide mismatch between the target ssDNA and the oligonucleotide probe, which allows detection of minor genetic variants in target genes in a bacterial population. Second, the shorter oligonucleotide probes allow independent testing of several species-specific regions of each gene, enabling effective coverage of the target sequence with more (but shorter) oligonucleotide probes. This reduces the probability of misidentification. Third, short oligonucleotides reduce the cost of chip production.

The redundancy of the testing (the number of spots representing each gene) is one way to reduce the risk of SE misidentification. While only one portion of each SE gene (the variable region shown in Fig. 1) is used for the analysis, leaving open the possibility of significant sequence variation in other parts of the genes, such variation is not common.

The main disadvantage of simultaneous PCR amplification of multiple targets is that different copy numbers of the genes in a cell result in different signal intensities on the array. This might be overcome by use of supplemental specific primers to improve the detection of underrepresented amplicons (Fig. 5 and 6).

Many of the known SEs have been discovered only in the last few years, including some, such as sep, that were discovered only through the sequencing of the S. aureus N315 genome (22). Given the genetic variability and the spread of the ent gene family, it is possible that there are other, as yet unknown, ent genes. However, the similarity in the conserved regions of these genes (Fig. 1) suggests that any additional gene family members will share those conserved sequences. Thus, it is possible that this assay might lead to the discovery of additional ent genes in new strains. The amplicons of the novel ent genes can be discernible as amplicons that hybridize to common spots but not to any ent gene-specific spots.

Multiple SE genes are commonly found in S. aureus strains (19, 20, 32). Among 198 S. aureus isolates implicated in S. aureus infections in France, 85.4% expressed multiple SEs (19). Our analysis of these data suggests that the majority (92%) contain multiple SEs, especially the egc cluster (seg, sei, sen, seo, and sem). Among the S. aureus isolates implicated in food-poisoning episodes in Japan, 93% expressed SEs, while only 72.2% of isolates from healthy people expressed SEs (32).

One explanation for the presence of multiple toxins in most strains is that these genes are often structurally linked. Several pathogenicity islands have been reported in S. aureus, including one encoding the toxic shock syndrome toxin (tst) and s- and sel-like proteins (13) and another encoding SE serotypes B, K, and Q (41). Others have reported pathogenicity islands containing the tst gene and an open reading frame with sequence similarity to those encoding SEs (25) and a region contains enterotoxins D and J (42). In addition, as noted above, a group of five toxin genes (seg, sei, sen, seo, and sem) is encoded by the enterotoxin gene cluster, egc (20). Interestingly, in the study of 198 clinical isolates by Jarraud et al. (19), half of the 14 strains that carried only a single enterotoxin gene had the sea gene, perhaps because it has been shown to be associated with a structurally unstable, possibly mobile, discrete genetic element (8) that is not part of the egc cluster.

In terms of the functionalities of SEs, multiple toxins with diverse spectra of activities may offer the pathogen versatility in terms of the host range. For example, it has been shown that different types of SEs have different emetic response activities in house musk shrews, although it was thought that there are no differences in the emetic response activities of SEA, SEB, SEC, SED, and SEE in humans and primates (18). In addition, S. aureus expression of specific ent genes may depend on the host tissue and may play a role in the adaptation of S. aureus to the host environment (4). Some have speculated that some cases of food poisoning result from the simultaneous expression of several enterotoxins in a single pathogenic S. aureus strain rather than from the expression of a single toxin.

However, it is unclear whether all the toxins are actually expressed and what the biological and clinical effects of multiple toxins might be. The method presented here can detect ent genes but does not determine whether the gene is expressed or whether the encoded protein is functional. The levels of correlation between the presence of genes that code for the production of SE (as determined by PCR) and the expression of these genes (as determined by enzyme-linked immunosorbent assay) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB (30). Thus, the actual presence of the toxin needs to be assessed by an immunological or activity assay.

In summary, the PCR-microarray method described here is a potentially powerful tool for the analysis of S. aureus strains. We used this method to test clinical isolates analyzed previously and found that these isolates frequently carry the genes for numerous toxins, including some of the newly discovered SEs. More studies need to be done to understand the biological regulation and the biological and clinical effects of multiple enterotoxins. Our method has great potential for application in high-throughput screening and accurate genotyping of ent genes, which are especially important in epidemiological studies.