J Med Microbiol 55 (2006), 273-277; DOI: 10.1099/jmm.0.46027-0
© 2006 Society for General Microbiology
ISSN 0022-2615
Development of a routine laboratory direct detection system of staphylococcal enterotoxin genes
Akifumi Nakayama1,
,
Akiko Okayama1,
Misao Hashida1,
Yasuzumi Yamamoto1,
Hisakatsu Takebe1,
Takashi Ohnaka2,
Tomoyuki Tanaka2 and
Shunsuke Imai1
1 Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara City, Nara 630-8131, Japan
2 Sakai City Institute of Public Health, 3-2-8 Kaichyo Higashi, Sakai City, Osaka 590-0953, Japan
Correspondence
Akifumi Nakayama
pyonchan{at}asint.jp
Received 28 January 2005
Accepted 21 October 2005
A novel direct detection system has been developed for eight staphylococcal enterotoxin (SE)-encoding genes (sea, seb, sec, sed, see, seg, seh and sei) in milk. Specific detection by real-time PCR was successful for all SE-encoding genes in the reference strains. Furthermore, a novel DNA-preparation method with good reproducibility [coefficients of variation 0·31, 0·99 and 1·21 % at 106, 104 and 102 c.f.u. (ml milk sample)–1, respectively] was developed to overcome PCR inhibition in the milk samples. The combination of this DNA-preparation method and real-time PCR resulted in high sensitivity [between 1·1x102 and 1·0x104 c.f.u. (ml milk sample)–1] and allowed the completion of the entire procedure within 4 h. Results of an evaluation of this method for the detection of SE-encoding genes using known outbreak milk samples produced results showing good correspondence with the reversed passive latex agglutination assay. In addition, this newly developed system can be applied to clinical samples such as faeces and vomit. Consequently, the system should be useful in the routine direct detection of SE-encoding genes in food-borne-poisoning samples.
Present address: Clinical Laboratory, Nara Prefectural Hospital, 1-30-1 Hiramatsu, Nara City, Nara 631-0846, Japan. 
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INTRODUCTION
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Food-borne poisoning by staphylococcal enterotoxins (SEs) is an important hazard. SEs, the emetic toxins produced by Staphylococcus aureus, have been divided into five serological types (SEA, SEB, SEC, SED and SEE) (Bergdoll, 1983). In recent years, new types of SEs (from SEG to SE1U) have been reported (Jarraud et al., 2001; Munson et al., 1998; Orwin et al., 2001; Ren et al., 1994; Su & Wong, 1995; Zhang et al., 1998; Sergeev et al., 2004; Omoe et al., 2003; Lina et al., 2004), but the emetic activity of several of these toxins has not been confirmed. However, various studies have shown that the newly described seg and sei genes are commonly found in food-borne and clinical isolates of S. aureus (Jarraud et al., 2001; McLauchlin et al., 2000; Omoe et al., 2002; Rosec & Giraud, 2002; Becker et al., 2003), suggesting that they have roles as pathogenic factors in staphylococcal food poisoning. Thus, the establishment of a method to detect eight enterotoxin genes (sea, seb, sec, sed, see, seg, seh and sei) is vital for the analysis of staphylococcal food poisoning. Recently, Letertre et al. (2003a) reported the detection of SE-encoding genes based on 5' nuclease multiplex PCR. However, this method cannot detect SE-encoding genes directly from samples because it requires a culturing process. Direct detection of pathogenic genes by PCR is hampered by inhibitors contained in the samples (Rossen et al., 1992; Wilson, 1997). Among the food samples, milk contains high concentrations of proteins and fats, which are major inhibitors of the PCR assay. In 2000, a large-scale and widespread outbreak of staphylococcal food poisoning caused by the ingestion of dairy products, including low-fat milk and yogurt beverages, occurred in Japan (Asao et al., 2003) affecting a total of 13 420 persons. This incident eloquently indicated the need for a rapid and direct detection method for routine laboratory analysis.
The aim of this study was to establish a system to directly detect eight SE-encoding genes in the food-borne-poisoning samples. To reach this objective, a novel DNA-preparation method and an real-time PCR method were developed, and these were evaluated using milk samples from a known outbreak (Asao et al., 2003) and clinical samples.
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METHODS
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Bacterial strains.
Five S. aureus strains (FRI-100, ATCC 14458, FRI-137, ATCC 23235 and FRI-326) were kindly provided by the Tokyo Metropolitan Institute of Public Health, Tokyo, Japan and were used as positive controls. The S. aureus FRI-137 strain has been reported to possess the following four enterotoxin genes (Letertre et al., 2003b): sec, seg, seh and sei. The following strains were used for negative controls: the staphylococci Staphylococcus epidermidis (ATCC 12228), Staphylococcus capitis (ATCC 35661) and Staphylococcus xylosus (ATCC 29971), and five common food-borne pathogens, Bacillus cereus (ATCC 11778), Campylobacter jejuni (ATCC 33291), Clostridium perfringens (ATCC 13124), Vibrio parahaemolyticus (ATCC 17802) and Yersinia enterocolitica (ATCC 9610).
Milk samples.
Pasteurized milk was purchased from the supermarket and stored at 4 °C until needed for the preparation of milk samples with known concentrations (106 c.f.u. ml–1) of S. aureus cells for the construction of standard curves. In addition, milk samples that gave rise to the staphylococcal food-poisoning outbreak (in the Kansai district of Japan from the end of June to the beginning of July 2000) described in Asao et al. (2003) were used for the evaluation of the direct detection system for SE-encoding genes. These milk samples were stored at –80 °C until needed.
Construction of standard curves.
S. aureus cells (positive control strains) cultured in tryptone soya broth at 37 °C were harvested at the exponential growth phase by centrifugation and resuspended in pasteurized milk. After DNA extraction from the milk samples, six serial dilutions of each DNA solution [106, 105, 104, 103, 102 or 101 c.f.u. (ml milk)–1] were made and analysed in triplicate for the production of a standard curve.
Identification of S. aureus strains from clinical samples.
S. aureus strains from clinical samples were checked by the Gram-staining test and the catalase production test (Palk, 1980). These strains were identified by coagulase production (Staphylase test; Oxoid), and by characterization using the VITEK system I (bioMérieux).
Detection of SEs by reversed passive latex agglutination (RPLA).
SEs, SEA, SEB, SEC, SED and SEE, were detected using the Enterotox-F RPLA kit (Denka Seiken), following the manufacturer's instructions, in the staphylococcal strains S. aureus, S. epidermidis, S. capitis and S. xylosus. SEs in milk samples were detected using a modified RPLA method (Asao et al., 2003).
Oligonucleotide primers and fluorescence-labelled probes.
Specific primer pairs, having identical annealing temperatures and working simultaneously under a single thermal-cycling condition, were designed using Primer Express software, version 2·0 (Applied Biosystems), based on the published sequences of SE-encoding genes (Betley & Mekalanos, 1988; Ranelli et al., 1985; Bohach & Schlievert, 1987; Bayles & Iandolo, 1989; Couch et al., 1988; Omoe et al., 2002). Fluorescence (fluorescein phosphoramidites)-labelled probes were designed with minor groove binder (MGB) to increase the specificity of probe hybridization and to enhance spectral performance. Specific MGB probes were synthesized by Applied Biosystems. The annealing temperatures of these fluorescence-labelled probes were approximately 6–11 °C higher than those of the PCR primers, allowing the probes to hybridize with the target region of the amplicon during the annealing/extension stage of each PCR cycle. The PCR primers and fluorescence-labelled probes used in this study are listed in Table 1
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Real-time PCR conditions.
Reaction mixtures (50 µl) contained 5 µl DNA template, 1xTaqMan buffer (Mg2+ free), 5 mM MgCl2, 200 µM dATP, dCTP and dGTP, 400 µM dUTP, 400 nM each specific primer pair, 200 nM of the appropriate TaqMan MGB probe, 0·5 U uracil-N-glycosylase, and 0·75 U AmpliTaq Gold DNA polymerase (Applied Biosystems). Separate reactions were carried out for each of the eight genes to retain amplification efficiency. Cycling conditions were: 50 °C for 2 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Reactions were carried out in a 96-well MicroAmp optical plate (Applied Biosystems) using the ABI Prism 7000 sequence detector (Applied Biosystems).
DNA-preparation method.
Through this study, we attempted to establish a direct detection system for SE-encoding genes in samples collected from cases of staphylococcal food poisoning. To achieve this goal, a new DNA-preparation method was developed. This consisted of the following three basic steps: step 1, alkaline treatment of samples to achieve cell lysis and inhibit nuclease activity; step 2, petroleum ether extraction to eliminate fats and proteins in the sample; step 3, application of a commercial kit for DNA purification and concentration. In this procedure, each milk sample (100 µl) was added to an equal volume 0·2 M sodium hydroxide, and the solution was incubated at 37 °C for 20 min. The alkaline treated sample (200 µl) was neutralized with 10 µl 3 M sodium acetate (pH 5·4), extracted with 1·0 ml petroleum ether, and then centrifuged at 13 000 g for 10 min at 25 °C. The aqueous phase was transferred to a fresh tube. Bacterial DNA was purified from the aqueous solution using a Wizard SV genomic DNA purification kit (Promega). Finally, DNA was eluted in 50 µl sterile distilled water and stored at –20 °C.
Cloning and sequencing of PCR products.
Real-time PCR products from reference strains were cloned into the pCRII-TOPO plasmid vector (Invitrogen) according to the manufacturer's instructions. DNA sequencing of the cloned amplification products was carried out by the Dragon Genomics Center (Takara Bio).
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RESULTS AND DISCUSSION
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Specificity of real-time PCR
The specificity of real-time PCR was tested for the positive and negative control strains. The eight SE-encoding genes (sea, seb, sec, sed, see, seg, seh and sei) were detected in the positive control strains and not in the negative control strains (Table 2). In addition, the DNA sequences of all real-time PCR products showed complete agreement with the sequences of the corresponding region of each SE-encoding gene (data not shown). Therefore, the eight pairs of primers designed in this study were determined to be completely specific for each SE-encoding gene.
Sensitivity and reproducibility of the direct detection system
Standard curves of threshold cycle (Ct) values versus the log10 of the c.f.u. values were plotted for milk samples inoculated with 106 c.f.u. S. aureus (ml milk)–1 for evaluation of the sensitivity and reproducibility of the combined newly developed DNA-preparation method and real-time PCR. All SE-encoding gene standard curves based on 10-fold dilutions of DNA showed a linear relationship between log10 c.f.u. and Ct value. The detection limit for each SE-encoding gene was between 1·1x102 (Ct=39·44) and 1·0x104 (Ct=37·92) c.f.u. (ml milk sample)–1 (Table 3), and the R2 values of the linear regressions were greater than 0·9736. Furthermore, the reproducibility of S. aureus (for strain ATCC 14458; n=5) cellular DNA-preparation from milk at levels of 106, 104 and 102 c.f.u. (ml milk)–1 had coefficients of variation of 0·31, 0·99 and 1·21 %, respectively. These results, good sensitivity and a high level of reproducibility, confirmed that this system is suitable for the direct detection of SE-encoding genes.
Direct detection of SE-encoding genes in milk samples
Since our system was targeted toward food poisoning samples, milk samples from a known staphylococcal outbreak were tested to demonstrate its applicability. Using previously available methods, SEA was detected in staphylococcal-outbreak milk samples by the modified RPLA method (Asao et al., 2003). For the modified RPLA method, a rotary evaporator was used to concentrate and extract the SE in the samples, and this method identified SEA as the only serotype detected in 20-fold concentrated milk samples. However, S. aureus strains were not isolated by the culture method. Thus, the detection of SE-encoding genes in the milk samples using our system was compared with RPLA results. Ten milk samples were examined for the presence of SE-encoding genes and SE proteins. As shown in Table 4, all milk samples producing positive results in RPLA reactions also showed positive real-time PCR results, while two milk samples (Sakai-11 and -29) were negative for both assays. These milk samples with negative results contained S. aureus at a considerably lower level or did not contain it at all. In addition, the real-time PCR results indicated that the positive milk samples contained not only sea, but also seg, seh and sei genes.
Application to clinical samples
We applied a newly developed system for the detection of SE-encoding genes to clinical samples. A total of twenty clinical samples (19 diarrhoea faeces and 1 vomit sample), from which S. aureus had been isolated, were used. Each clinical sample was suspended with an equal volume of PBS, pH 7·0, and then subjected to our direct PCR system. S. aureus strains (5 colonies) isolated from each clinical sample were also examined for the existence of SE-encoding genes by real-time PCR. The real-time PCR results of the direct detection showed good correspondence with results of previously isolated S. aureus strains from 20 clinical samples (Table 5). SE-encoding genes were detected, using the direct PCR system, in all 11 positive samples, and in none of the 9 negative samples. Furthermore, the direct PCR system also identified more SE-encoding genes than did the isolation method for three of the samples, indicating the possibility that other enterotoxigenic S. aureus strains without viability were present in the clinical samples.
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Table 5. Comparison of SE-encoding genes detected in clinical samples using previously isolated strains and using the direct detection method
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In conclusion, we have described the development of a system for the direct detection of SE-encoding genes in milk samples. The system is composed of a novel DNA-preparation method and real-time PCR, and showed good specificity and sensitivity for the detection of SE-encoding genes. In addition, the DNA-preparation method had a high level of reproducibility based on the determined coefficients of variation. Furthermore, this method was also applicable to clinical samples with high-levels of PCR inhibitors. Ramesh et al. (2002) reported a method of DNA extraction from milk for the detection of S. aureus and Y. enterocolitica by PCR. Their method successfully overcame the inhibition on the PCR assay. Because it detects a staphylococcal nuclease gene as a target of PCR, the sensitivity of the PCR is higher than in our method. However, the DNA-preparation step of their method is time consuming. Our newly developed direct detection system allows the completion of the entire procedure (DNA preparation and real-time PCR) within 4 h. Thus, our system is convenient for routine laboratory applications, especially in the investigation of food poisoning outbreaks caused by S. aureus.
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ACKNOWLEDGEMENTS
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We thank Dr Keizo Yamamoto (Department of Chemistry, Nara Medical University, Nara, Japan) for valuable suggestions in this study.
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REFERENCES
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Asao, T., Kumeda, Y., Kawai, T., Shibata, T., Oda, H., Haruki, K., Nakazawa, H. & Kozaki, S. (2003). An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk. Epidemiol Infect 130, 33–40.[CrossRef][Medline]
Bayles, K. W. & Iandolo, J. J. (1989). Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin D. J Bacteriol 171, 4799–4806.[Abstract/Free Full Text]
Becker, K., Friedrich, A. W., Lubritz, G., Weilert, M., Peters, G. & von Eiff, C. (2003). Prevalence of genes coding for pyrogenic toxin superantigens and exfoliative toxins in blood and nasal strains of Staphylococcus aureus. J Clin Microbiol 41, 1434–1439.[Abstract/Free Full Text]
Bergdoll, M. S. (1983). Enterotoxins. In Staphylococci and Staphylococcal Infections, pp. 559–598. Edited by C. S. F. Easton & C. Adlam. London: Academic Press.
Betley, M. J. & Mekalanos, J. J. (1988). Nucleotide sequence of the type A staphylococcal enterotoxin gene. J Bacteriol 170, 34–41.[Abstract/Free Full Text]
Bohach, G. A. & Schlievert, P. M. (1987). Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins. Mol Gen Genet 209, 15–20.[CrossRef][Medline]
Couch, J. L., Soltis, M. T. & Betley, M. J. (1988). Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene. J Bacteriol 170, 2954–2960.[Abstract/Free Full Text]
Jarraud, S., Peyrat, M. A., Lim, A. & 7 other authors (2001). egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J Immunol 166, 669–677.[Abstract/Free Full Text]
Letertre, C., Perelle, S., Dilasser, F. & Fach, P. (2003a). A strategy based on 5' nuclease multiplex PCR to detect enterotoxin genes sea to sej of Staphylococcus aureus. Mol Cell Probes 17, 227–235.[CrossRef][Medline]
Letertre, C., Perelle, S., Dilasser, F. & Fach, P. (2003b). Detection and genotyping by real-time PCR of the staphylococcal enterotoxin genes sea to sej. Mol Cell Probes 17, 139–147.[CrossRef][Medline]
Lina, G., Bohach, G. A., Nair, S. P., Hiramatsu, K., Jouvin-Marche, E. & Mariuzza, R. (2004). Standard nomenclature for the superantigens expressed by staphylococcus. J Infect Dis 189, 2334–2336.[CrossRef][Medline]
McLauchlin, J., Narayanan, G. L., Mithani, V. & O'Neill, G. (2000). The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction. J Food Prot 63, 479–488.[Medline]
Munson, S. H., Tremaine, M. T., Betley, M. J. & Welch, R. A. (1998). Identification and characterization of staphylococcal enterotoxin type G and I from Staphylococcus aureus. Infect Immun 66, 3337–3348.[Abstract/Free Full Text]
Omoe, K., Ishikawa, M., Shimoda, Y., Hu, D.-L., Ueda, S. & Shinagawa, K. (2002). Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, or sei genes. J Clin Microbiol 40, 857–862.[Abstract/Free Full Text]
Omoe, K., Hu, D. L., Takahashi-Omoe, H., Nakane, A. & Shinagawa, K. (2003). Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids. Infect Immun 71, 6088–6094.[Abstract/Free Full Text]
Orwin, P. M., Leung, D. Y. M., Donahue, H. L., Novick, R. P. & Schlievert, P. M. (2001). Biochemical and biological properties of staphylococcal enterotoxin K. Infect Immun 69, 360–366.[Abstract/Free Full Text]
Palk, G. (1980). Reagents, strains and miscellaneous test procedures. In Manual of Clinical Microbiology, 3rd edn, pp. 1000–1024. Edited by E. Lennette, A. Balows, W. Hausler Jr & J. Truant. Washington, DC: American Society for Microbiology.
Ramesh, A., Padmapriya, B. P., Chandrashekar, A. & Varadaraj, M. C. (2002). Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples. Mol Cell Probes 16, 307–314.[CrossRef][Medline]
Ranelli, D. M., Jones, C. L., Johns, M. B., Mussey, G. J. & Khan, S. A. (1985). Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus. Proc Natl Acad Sci U S A 82, 5850–5854.[Abstract/Free Full Text]
Ren, K., Bannan, J. D., Pancholi, V., Cheung, A. L., Robbins, J. C., Fischetti, V. A. & Zabriskie, J. B. (1994). Characterization and biological properties of a new staphylococcal enterotoxin. J Exp Med 180, 1675–1683.[Abstract/Free Full Text]
Rosec, J. P. & Giraud, O. (2002). Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. Int J Food Microbiol 77, 61–70.[CrossRef][Medline]
Rossen, L., Norskov, P., Holmstrom, K. & Rasmussen, O. F. (1992). Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol 17, 37–45.[CrossRef][Medline]
Sergeev, N., Volokhov, D., Chizhikov, V. & Rasooly, A. (2004). Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay. J Clin Microbiol 42, 2134–2143.[Abstract/Free Full Text]
Su, Y. C. & Wong, A. C. L. (1995). Identification and purification of a new staphylococcal enterotoxin, H. Appl Environ Microbiol 61, 1438–1443.[Abstract]
Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63, 3741–3751.[Medline]
Zhang, S., Iandolo, J. J. & Stewart, G. C. (1998). The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiol Lett 168, 227–233.[CrossRef][Medline]
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INTRODUCTION
METHODS
