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1 ,3 Department of Biological Sciences, Faculty of Science1 and Department of Pathology and Microbiology, Faculty of Medicine3 , University of Jordan, Amman, Jordan
2 National Center for Agricultural Research and Technology Transfer, Amman, Jordan
Correspondence
Salwa M. Bdour
bsalwa{at}ju.edu.jo
Received 2 June 2005
Accepted 10 October 2005
Abbreviations: RAPD, randomly amplified polymorphic DNA; SE, staphylococcal enterotoxin.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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The enterotoxins of S. aureus can be detected by their biological activity, by immunoassays and by PCR (McLauchlin et al., 2000). The prevalence of enterotoxigenic clinical S. aureus isolates has been reported in different countries by many investigators (Mehrotra et al., 2000; Fueyo et al., 2001; Becker et al., 2003). There are no published reports on the prevalence of these genes in Jordanian S. aureus isolates.
Accurate and rapid typing of S. aureus strains is crucial to the control of infectious strains. Numerous typing methods have been described (Frénay et al., 1994; Kluytmans et al., 1995; Yoshida et al., 1997; van Leeuwen et al., 2003); among these is randomly amplified polymorphic DNA (RAPD) analysis, which has been found to be a simple, rapid and effective method for genotyping of S. aureus (Tambic et al., 1997).
The purpose of this study was to investigate (i) the prevalence of the classical enterotoxin genes in Jordanian clinical isolates of S. aureus, (ii) the genetic variation among these isolates using RAPD analysis and (iii) the genotype specificity of the classical enterotoxin genes.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Genomic DNA. Genomic DNA was extracted from overnight cultures of S. aureus using the Wizard Genomic DNA Purification kit (Promega). The procedure was identical to that recommended by the manufacturer, except that the pelleted bacterial cells were first treated with 60 µl lysozyme (10 mg ml–1; ICN Biomedicals) and 9 µl lysostaphin (1 mg ml–1; Sigma) for 1 h at 37 °C. The preparations were analysed on a 0·7 % agarose gel and the quantity and quality of DNA were determined spectrophotometrically (Sambrook et al., 1989). The amount of DNA was adjusted to the required concentration for each PCR for detection of enterotoxin genes and for genotyping of S. aureus isolates.
Detection of enterotoxin genes by PCR. Genomic DNA (50–100 ng) of S. aureus strains was amplified in two sets of multiplex PCR as reported by Rosec & Gigaud (2002). Set A contained 3 ng of each sea, sed and see primer µl–1, while set B contained 3 ng of each seb and sec primer µl–1. Amplification with these primers gave rise to PCR products of 544, 416, 257, 334 and 170 bp for sea, seb, sec, sed and see, respectively. A positive PCR control containing DNA of the reference strains and a negative PCR blank control with nuclease-free water instead of genomic DNA were included with each set of five reactions.
RAPD-PCR typing. Four random-sequence primers were used in four separate RAPD-PCR tests for typing of S. aureus isolates. Primers S (5'-TCACGATGCA-3') and C (5'-AGGGAACGAG-3') (Fueyo et al., 2001) and primer OPA13 (5'-CAGCACCCAC-3') (Byun et al., 1997) were selected because they previously showed good discriminatory power in RAPD analysis of S. aureus (Fueyo et al., 2001; Byun et al., 1997). Primer OPA09 (5'-GGGTAACGCC-3') (Operon Technologies) was used after optimization of PCR conditions for ultimate discriminatory power.
RAPD-PCR with 0·5 µM primer S or C and 100 ng genomic DNA was performed according to Fueyo et al. (2001), and with 0·1 µM primer OPA13 and 50 ng DNA was performed according to Byun et al. (1997). After optimization of PCR components and conditions, RAPD-PCR with OPA09 was carried out in a 25 µl reaction mixture containing 12·5 µl PCR Master Mix (Promega), 10 pmol primer, 3 U Taq DNA polymerase, 3·5 mM MgCl2, 10 ng DNA template and nuclease-free water. Amplification conditions consisted of denaturation at 94 °C for 60 s and 35 cycles of denaturation at 94 °C for 35 s, annealing at 33 °C for 30 s and extension at 72 °C for 65 s. PCR products were detected in 1 % agarose gel.
Computer analysis of RAPD data. RAPD banding patterns of the 100 isolates of S. aureus were examined and the size of bands was estimated by using the standard curve for DNA marker fragments in each gel (Sambrook et al., 1989). Bands were scored, with the data coded as a factor of 1 or 0, representing the presence or absence of bands, respectively. Banding patterns of the summed results for the four primers were used for cluster analysis in the RAPD assay. A similarity matrix among S. aureus isolates was produced by using the Jaccard coefficient (Jaccard, 1908) and a dendrogram showing the genetic relatedness among the isolates was constructed from the resulting data using SPSS version 10. The cut-off for the dendrogram was selected based on the average of mean similarity matrix.
Statistical analysis.
A 
2 test was used to study the correlation between the prevalence of the enterotoxin genes and the genotypes of S. aureus isolates. A value of P<0·05 was considered statistically significant (Levin & Rubin, 1991).
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
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). All 100 polymorphic bands produced were used for cluster analysis. The genetic similarity indices based on Jaccard coefficients ranged from 0·1 to 1·0. This wide range of similarity indices indicated a high level of DNA polymorphism among the S. aureus isolates. The average mean similarity index was 0·47, indicating that the isolates shared 47 % of their RAPD fragments.
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shows a dendrogram constructed on the basis of similarity index among S. aureus isolates using the four RAPD primers. A 47 % similarity cut-off value gave 13 major clusters (RAPD genotypes) (I–XIII) and seven subclusters (RAPD subtypes) including genetically related isolates. The majority of S. aureus isolates (43) belonged to genotype I (range of similarity 0·46–1·00). The others were distributed as follows: 26 isolates in genotype VI (range of similarity 0·30–1·00), 17 isolates in genotype V (range of similarity 0·41–0·85), three isolates in genotype X (range of similarity 0·6–0·8), two isolates in genotype VII (similarity level 0·74) and two isolates in genotype VIII (similarity level 0·6). Seven isolates formed single clusters (genotypes) (II, III, IV, IX, XI, XII and XIII). On the other hand, genotype VI included three subtypes: VIa at a 0·63 similarity level and VIb and VIc at a 0·55 similarity level. Genotype V included four subtypes: Va, Vb and Vc at a 0·60 similarity level and Vd at a 0·56 similarity level.
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shows the distribution of the enterotoxin genes among S. aureus genotypes. The sea gene was carried either singly or in combination with the sec gene by some isolates of genotypes I, V (subtypes Vb, Vc and Vd), VI, IX and X. The sec gene was harboured by some isolates of genotypes V (subtype Vb and Vd), VI (subtype VIa) and X. On the other hand, isolates of the single genotypes (II, III, IV, XI, XII and XIII), genotypes VII and VIII and subtypes Va, VIb and VIc were negative for the toxin genes.
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2 test showed that the overall presence of the enterotoxin genes was independent of genotype. There was no significant difference (P<0·05) in the prevalence of the tested SE genes in the different S. aureus genotypes. |
DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, the sea gene dominated over the sec gene. Similar results have been reported in Germany (Becker et al., 2003) and Canada (Mehrotra et al., 2000) among isolates of human origin. However, other investigators have reported that the sec gene is the most frequent among strains of bovine origin in Europe and the USA (Valle et al., 1990; Sharma et al., 2000; Larsen et al., 2002).
It is important to recognize that the presence of the sea and sec genes in Jordanian S. aureus isolates does not necessarily indicate the ability of these isolates to produce intact and biologically active toxin or to produce sufficient toxin to induce disease. Detection of these genes by PCR may indicate their potential to cause disease. Fueyo et al. (2001) reported that the detection of the classical enterotoxin genes in Spanish S. aureus isolates by PCR was fully concurrent with the production of toxin by these isolates. In contrast, Sharma et al. (2000) reported that toxin genes were detected in toxin-non-producing strains of S. aureus, due either to low-level production of enterotoxin or to mutations in the coding region or in a regulatory region(s) (Sharma et al., 2000).
Typing by RAPD revealed that the 100 clinical S. aureus isolates were genetically diverse and comprised a heterogeneous population with 13 genotypes at a 47 % similarity level. Genotype I appeared to be predominant. The presence of sea and sec genes was not genotype specific. Genotype I included only sea-positive isolates (Table 1
), while genotype V included a great diversity of sea- and sec-positive isolates. More importantly, pairs of identical isolates with a similarity index of 1·00 (e.g. pairs of isolates numbered 11 and 65, 38 and 63, and 67 and 68 of genotype I; Fig. 3) included sea- and sec-positive and -negative isolates. This result provides evidence that loss and/or acquisition of enterotoxin genes occurs within the same genetic background and that the presence of these genes is not genotype specific. In agreement with our results, Fueyo et al. (2001) reported that some SE-positive and some SE-negative strains generated identical RAPD banding profiles, suggesting that SE-positive strains do not belong to a specific genetic class. In addition, Araki et al. (2002) showed that there was no association between RAPD genotypes and the presence of toxin genes.
Horizontal movement of sea or sec genes could occur among isolates of different genotypes. This movement appeared to be restricted to five genotypes (I, V, VI, IX and X) (Table 1) and could be extended to other genotypes and involve isolates negative for the toxin genes. The horizontal transfer of enterotoxin genes is mostly mediated by generalized transduction (Moore & Lindsay, 2001). The fact that genotypes I and V included most sea- and sec-positive S. aureus isolates may indicate their occurrence as epidemic isolates in the Jordanian community and hospitals.
In conclusion, the present investigation demonstrated that the sea gene was the predominant enterotoxin gene in these genetically diverse Jordanian clinical isolates and that the presence of these genes was not genotype specific.
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ACKNOWLEDGEMENTS |
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