Prevalence of seg, seh and sei Genes among Clinical and Nasal Staphylococcus aureus Isolates in Palestine

Aims: To investigate the presence of the staphylococcal enterotoxin genes seg, seh and sei among clinical and nasal isolates. Place and Duration of Study: Department of Biology and Biotechnology, An-Najah N. University, Palestine, in 2011. Methodology: A total 124 S. aureus isolates were collected, forty three were nasal and 81 were clinical isolates. PCR technique was used to detect enterotoxin genes seg, seh and sei , mecA gene and analysis of SCC mec types. Enterotoxigenic strains were also typed using coagulase typing kit. Results: Fifty two (41.9%) isolates were positive for one or more of these enterotoxin genes. The prevalence of toxin genes among S. aureus isolated from nasal swabs 25/43 (58.1%) was higher than those isolated from clinical samples 27/81 (33.3%). Combination of the toxin genes was noted only in MSSA isolate from both nasal swabs and clinical samples. Distribution of toxin genes in MSSA isolates was higher (49.5%) than those in MRSA isolates (21.2%). SCC mec typing showed that the MRSA enterotoxigenic strain were belonged to types II, III and IVa. MRSA strains were found to belong to coagulase serotypes II, III and VII, while MSSA strains were belonged to serotypes II-VII. In nasal samples, 16/25 (64.0%) of enterotoxigenic strains showed the genotype seg + /sei + , while in clinical samples 1/27 (3.7%), 1/27 (3.7%) and 3/27 (11.1%) of enterotoxigenic strains showed the genotypes seg + /seh + , seg + /sei + and seg + /seh + /sei + , respectively. This study showed that the majority of the isolates 42/124 (33.9%) were seg + , while none of nasal strains harbored seh gene. Conclusion: The prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples, as well as the prevalence of the same genes in MSSA differed significantly from those in MRSA. In addition, S. aureus isolates from clinical and nasal swabs could serve as a possible reservoir of newly described seg , seh and sei genes.

serotypes II, III and VII, while MSSA strains were belonged to serotypes II-VII. In nasal samples, 16/25 (64.0%) of enterotoxigenic strains showed the genotype seg + /sei + , while in clinical samples 1/27 (3.7%), 1/27 (3.7%) and 3/27 (11.1%) of enterotoxigenic strains showed the genotypes seg + /seh + , seg + /sei + and seg + /seh + /sei + , respectively. This study showed that the majority of the isolates 42/124 (33.9%) were seg + , while none of nasal strains harbored seh gene. Conclusion: The prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples, as well as the prevalence of the same genes in MSSA differed significantly from those in MRSA. In addition, S. aureus isolates from clinical and nasal swabs could serve as a possible reservoir of newly described seg, seh and sei genes.

INTRODUCTION
Staphylococcus aureus is considered one of the most important pathogen to humans and animals. In human, many different strains of S. aureus are capable of causing a wide variety of diseases ranging from skin and tissue infections, toxin-mediated diseases, pneumonia, and bacteremia [1]. Staphylococcus aureus infections can be acquired through both hospital and community settings. However, many of these infections are difficult to treat because of evolved resistance to antimicrobial drugs. The emergence of methicillin-resistant S. aureus (MRSA) strains and other antimicrobial agents has become a major concern, especially in the hospital environment, because of increased mortality due to systemic MRSA infections [2]. About 30% of individuals carry this organism in the anterior nares at any given time [3], and more than 70% of isolates recovered from healthy population were enterotoxigenic [4].
There are several types of virulence factors produced by this pathogen, which contribute in different ways to pathogenicity. These factors include degradative enzymes, surfaceassociated factors and superantigenic toxins [5]. Staphylococcal superantigens (SAgs) including staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) were originally identified in S. aureus. Several SEs were designated as SE-like (SEl) since they either lack emetic properties or their emetic activities have not been examined. More than 20 types of SEs/SEls which are usually subdivided into classical SEs (SEA-SEE), newly described SEs (SEG-SEI and SER-SET) and SEls (SElJ-SElQ, SElU and SElV) [6,7]. These toxins are encoded by accessory mobile genetic elements, including prophages, plasmids, chromosomal pathogenicity islands (SaPIs or vSa), or by genes located next to the staphylococcal cassette chromosome (SCC) implicated in methicillin resistance [6,8,9].
Staphylococcal enterotoxins are a group of toxins resistant to heat treatment, low pH and proteolytic digestive enzymes such as pepsin, with low molecular mass proteins of approximately 27-31 kDa, similar in composition and biological activity but differ in antigenicity [6,10,11]. Products of seg, seh and sei genes showed emetic activity with an animal model [12], but it is not yet clear if seg and sei genes are responsible for food poisoning in humans. There is limited information about the prevalence of the recently identified enterotoxins in human population in comparison to the classical enterotoxins.
Enterotoxin-producing S. aureus plays an important role in food intoxications. Food handlers carrying enterotoxin-producing S. aureus in their noses or on their hands are considered as the major source of food contamination, via manual contact or through respiratory secretions. Staphylococcal food poisoning, caused by ingestion of enterotoxins is characterized by emesis and in some cases is accompanied by gastro-enteritis.
To the best of our knowledge, there are no published reports about the occurrence of these newly described staphylococcal enterotoxin genes in Palestinian clinical and nasal S. aureus isolates. The purpose of the present study was to investigate the presence of the SE genes seg, seh and sei in sets of MSSA and MRSA isolates collected from different clinical samples and nasal swabs from healthy students during 2011.

Sample Collection and Identification
A total of 124 S. aureus isolates were collected in 2011, in which 43 were nasal swab isolates and 81 were clinical isolates. Nasal swabs were obtained from 95 healthy students, Dept. of Biology and Biotechnology, An-Najah National University-Palestine, including 15 males and 80 females. Their ages varied between 19 and 24 and all declared that have not received antibiotics recently. Nasal samples were collected from students with swab rubbed inside one nostril followed by the other, yielding a paired swab sample. After sampling, swabs were immediately transferred into 5 mL nutrient broth and incubated for 18-24 h at 37ºC. Thereafter, 100 μL of the nutrient broth were sub cultured for identification. Clinical isolates were obtained from three different health centers in northern Palestine in the same time period of the study. The patients were 35 females and 46 males, between 5 and 70 years old. S. aureus was isolated from all the pathological samples (36 urine samples, 7 semen samples, 8 blood samples, 15 wound samples and 15 diabetic foot samples). Replicate isolates from the same patient were excluded. Identification of S. aureus was confirmed on the basis of Gram stain, catalase, culture properties on mannitol salt agar, detection of hemolysis on blood agar, and coagulase reaction.

Antimicrobial Susceptibility Testing
S. aureus strains were tested for Methicillin resistance using the disk diffusion method [13]. Methicillin (5 µg) discs (Oxoid) were used in this test. Inhibition zones were determined in accordance with procedures of the Clinical and Laboratory Standards Institute (formerly NCCLS) [14]. Isolates were categorized as susceptible if inhibition zones were ≥14 mm. Otherwise, the isolates were considered as resistant. All methicilline resistant S. aureus strains identified by disk diffusion method were also confirmed by the presence of gene mecA using PCR as described previously [15]. Methicillin-resistant control strains from our department collection and methicillin-susceptible reference (S. aureus ATCC 25923) were used in this study.

DNA Extraction
S. aureus genome was prepared for PCR by boiling and cooling method. Briefly, cells were scraped off an overnight nutrient agar plate with a sterile loop, washed twice in 1 ml of 1X Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA [pH 7.5]), re suspended in 0.5 ml of sterile distilled H 2 O, and boiled for 10-15 min. The cells then were incubated on ice for 10 min. The debris was pelleted by centrifugation at 11,500 X g for 5 min, and the supernatant containing the released DNA was transferred into a new Eppendorf tube and stored at -20ºC for used.

PCR amplification of mecA
The mecA gene was detected by PCR with specific primers: mecA1 (5'-TGG CTA TCG TGT CAC AAT CG-3') and mecA2 (5'-CTG GAA CTT GTT GAG CAG AG-3') were used for amplification of a fragment of 310 bp [15]. The PCR reaction mix with a final volume of 50 μL, was composed of 5 μL of extracted DNA, 0.4 μM of each primer, 0.2 mM of each dNTP, 1.8 mM of MgCl 2 , 1X PCR buffer and1.5 U of Taq DNA polymerase. The amplification was carried out using the thermal cycler (Mastercycler personal, Eppendorf, Germany), after initial denaturation stage at 92ºC for 1 min, 40 cycles of amplification were preformed as follows: denaturation at 92ºC for 20 s, annealing at 58ºC for 20 s, and extension at 72ºC for 20s, with increment of 2s per cycle for the denaturation and extension segments. At the end of the cycles, the reaction mixture was achieved with final extension at 72ºC for 5 min. After amplification PCR products were analyzed by electrophoresis on 2% (w/v) agarose gel. Methicillin-resistant control strains from our department collection were used in this study.

Multiplex PCR assay for SCCmec typing
The primer sets used for SSCmec typing were described previously [16]. Each PCR reaction mix (50 μL) contained 1X PCR buffer, 1.5 U Taq DNA polymerase, 3 mM MgCl 2, 5 μL primer mix (2 mM in TE buffer for each primer), 0.2 mM dNTPs and 3 μL DNA template. DNA amplification was carried out using the thermal cycler (Mastercycler personal, Eppendorf, Germany) according to the following thermal conditions: initial denaturation for 5 min at 94ºC followed by 30 cycles of initial denaturation 94ºC for 30 s, 57ºC for 1.5 min and 72ºC for 1.5 min ending with a final extension step at 72ºC for 5 min, and followed by a holding step at 4ºC. The PCR products (10 μL) were analyzed by electrophoresis on 2% agarose gel. The SCCmec type was determined on the basis of the band pattern obtained. Each individual PCR yielded the fragments of expected size: i.e. 613, 287, 243, 776, 1000, 677, 1242, 663 and 325 bp for subtypes I, II, III, IVa, IVb, IVc, IVd, IVh and V, respectively. Control strains for different SCCmec types from our department collection were used in this study.

Detection of the toxin genes
The primer sets used for the detection of seg, seh and sei genes were described previously [17]. Each PCR reaction mix (50 μL) contained 1X PCR buffer, 1.5 U Taq DNA polymerase, 2 mM MgCl 2, 0.4 μM of each primer, 0.2 mM dNTPs and 3 μL DNA template. DNA amplification was carried out using the thermal cycler (Mastercycler personal, Eppendorf, Germany) according to the following thermal conditions: initial denaturation for 1 min at 94ºC followed by 30 cycles at 94ºC for 30s for denaturation, and extension at 72ºC for 30s. Different annealing temperatures were used 55ºC, 46.4ºC and 50ºC for 30 s for seg, seh and sei genes, respectively. Final extension was carried out at 72ºC for 5 minutes. After amplification PCR products were analyzed by electrophoresis on 2% agarose gel. Amplification of non template controls was attempted with each reaction to determine if DNA contamination occurred. The mixture of genomic DNA from S. aureus reference strains (ATCC 51811 for seh and S. aureus ATCC 25923 for seg and sei genes) were used for PCR of each set as a positive control.

Coagulase Typing
Coagulase serotyping was performed with the coagulase Seiken typing kit (Denka-Seiken, Japan) according to the manufacturer's instructions.

Statistical Analysis
A χ 2 test was used to study the correlation between the prevalence of the enterotoxin genes in different groups of S. aureus isolates. Statistical analysis was done using statistical package, SPSS windows version 15. A value of P<0.05 was considered statistically significant.

RESULTS
Our results showed that all the MRSA isolates detected by disk diffusion method were displayed positive amplifications of the mecA gene. Detection of enterotoxin genes by PCR showed that fifty two (41.9%) of S. aureus isolates were positive for one or more of these genes. The prevalence of toxin genes among S. aureus isolated from nasal swabs 25/43 (58.1%) was higher than those isolated from clinical samples 27/81 (33.3%). Combination of the toxin genes was noted only in MSSA isolates from both nasal swabs and clinical samples. The prevalence of toxin genes in MSSA isolates was higher (49.5%) than those in MRSA isolates (21.2%). In nasal swab samples, 16 Table 1. The X 2 test showed that the prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples and the P value was 0.008, while the prevalence of the same genes in MSSA differed significantly from those in MRSA with a P value was 0.005. a Statistically significant association after analysis (P < 0.05), the P value was 0.008. b Statistically significant association after analysis (P < 0.05). the P value was 0.005 The X 2 test also showed that the overall presence of the enterotoxin genes was independent of the coagulase serotypes. The SCCmec typing showed that the MRSA enterotoxigenic strains isolated from both clinical and nasal swab samples were belonged to type II, III and IVa. Coagulase typing showed that all the MRSA strains were found to belong to coagulase serotypes II, III and VII, while MSSA strains were belonged to serotypes II-VII and 2 strains were not detected. Coagulase type, SCCmec type of enterotoxigenic S. aureus isolated from nasal and different clinical sources are presented in Table 2.

DISCUSSION
Staphylococcal super antigens are virulence factors associated with S. aureus, which have pivotal roles on colonization, modulating the host immune response and also ability to cause food poisoning in humans [5,7,18]. Results from different reports have shown the high incidence of genes encoding new SEs and SEls among food-borne S. aureus [6]. Of these new enterotoxins, only seh + strains have clearly been involved in staphylococcal food poisoning outbreaks. Our results showed that all S. aureus isolated from nasal swabs did not carry she, meaning that these strains had not ability to cause food poisoning in humans. However, food poisoning outbreaks due to seg + , seh + and sei + S. aureus without producing any of the classical SEs have been described previously [4,8,19,20]. It was pointed out that, the presence of seg and sei genes did not mean the production of enough quantities of SEG and SEI to cause the food poisoning [4]. It is possible that the strains harboring new enterotoxin genes specially seg and sei may coexist with bacterial pathogens other than S. aureus that made tracing the origins of food poisoning cases more complicated [20], or the presence of multiple SE genes in S. aureus makes it less clear whether any single toxin is responsible for staphylococcal food poisoning [18]. It was shown that new enterotoxin genes mainly seg, sei, sek and seq were found in isolates from patients rather than strains from food samples and food poisoning [21]. Accordingly, these superantigens may contribute to enhance the persistence of infection. However, as their role as virulence factors in infection is still purely speculative, the main interest of the enterotoxins resides in their ability to cause food poisoning. Clinical S. aureus strains involved in staphylococcal toxic shock syndrome and scarlet fever without producing classical SE's or toxic shock syndrome toxin 1, leading to the suggestion that SEG or SEI toxins caused these diseases [22].
In Palestine, the prevalence of classical staphylococcal enterotoxin genes was determined from human clinical samples and it has ranged 41.2% to 63.3% [23,24]. In this study, up to 41.9% of S. aureus strains isolated from nasal swabs of healthy students and different clinical isolates carried seg, seh and/or sei genes. The prevalence of enterotoxigenic S. aureus isolates carried one or more of seg, seh and/or sei genes from different reports has ranged from 30% to 68.6 % [1,4,5,12,17,[25][26][27][28][29][30]. The prevalence of seg, seh and sei genes in this study was 33.9%, 8.1% and 19.4%, respectively. The prevalence of seg gene in previous reports has ranged from 8.6% to 63.9%, seh from 0% to 54% and sei from 11.5% to 62.8% [1,4,5,12,17,20,[25][26][27][28][29][30]. This variation in the prevalence of these genes between these studies might be attributable to different factors such as the geographical difference, which may be further affected by the different ecological origins of the isolated strains (food, humans and animals), type of sample (blood, urine, nasal swab, milk, meat), number of samples and methodology.
This study showed that seg gene was the most commonly detected among positive enteroxigenic strains, then sei gene, while seh gene was the lowest. These results were in contrast to previous study [4], which reported that seh gene was the most frequently detected among positive enteroxigenic strains isolated from humans with food poisoning and from healthy humans. Our results were consistent with a previous report [27], which showed that seg gene was the most frequently detected among clinical strains, then sei gene, while seh gene was the lowest. Other previous reports showed that the distribution of seg and sei genes from different clinical isolates had the same prevalence or approximately equal, while seh gene detected in very low or absent [26,28,32,[35][36][37].
Our results showed that seg and sei genes were detected more frequently in MSSA. These results were inconsistent with the previous reports which showed that seg and sei genes were detected more frequently in MRSA isolates [28,32]. Recent report showed no correlation between number of SE genes and degree of antimicrobial resistance [37]. MRSA is produced when MSSA acquires a mobile genetic element, SCCmec. Toxin-producing MSSA may also change the pathogenicity of established MRSA by the transfer of virulence factors via mobile genetic elements or plasmids.
Coagulase serotyping revealed that the 45 enterotoxigenic S. aureus isolates were serologically diverse and comprised a heterogeneous population with 6 coagulase serotypes. Although the identified S. aureus coagulase serotypes harbored the tested SE genes either singly or in combination, the X 2 test showed that the overall presence of the enterotoxin genes was independent of the coagulase serotypes. Horizontal transfer of these genes could occur among isolates of different coagulase serotypes.

CONCLUSION
Our findings in this pilot study support the conjecture that S. aureus isolates collected from both clinical and nasal swabs could serve as a possible reservoir of newly described seg, seh and sei genes. The prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples, as well as the prevalence of the same genes in MSSA differed significantly from those in MRSA. Further studies are needed to confirm the expression of these new enterotoxin genes by clinical strains of S. aureus, and to asses their significance in diseases. Based on our knowledge, the present study represents the first investigation carried out in Palestine regarding the relationship among the origin (nasal swabs or clinical strains), enterotoxigenicity and Methicilline resistant or sensitive S. aureus isolates.