Staphylococcus aureus (S. aureus) is a major pathogen of increasing importance due to the rise in antibiotic resistance [1] . It is often found as a commensal associated with skin, skin glands, and mucous membranes, mainly in the nose of healthy individuals [2] . Approximately 20% - 30% of the general populations are S. aureus carriers [3] .

S. aureus is associated with severe community and hospital acquired diseases and has for a long time been considered as a major problem of public health [4] . Many extracellular proteins and toxins are produced which probably contribute to the virulence of this organism [5] . S. aureus can produce exotoxins including exfoliative toxins, leukocidins, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (SEs) [6] . The spectrum of staphylococcal diseases ranges from pimples and furuncles to toxic shock syndrome and sepsis, most of which depend on many virulence factors. In contrast, some diseases, such as staphylococcal food poisoning, rely on only one type of virulence factors: the staphylococcal enterotoxins (SEs) [4] .

Staphylococcal food poisoning (SFP) is a very common, however, a rarely reported, type of food poisoning. Symptoms of SFP include rapid onset (within 1 - 6 h) of nausea, vomiting, diarrhea and abdominal cramps [7] . These symptoms subside within 24 - 48 h but the disease may remain for 7 - 10 days [8] . The staphylococcal enterotoxins (SEs), produced by S. aureus, are known as etiologic agents of food poisoning in man, and as potent immunologic superantigens [9] that stimulate non-specific T cell proliferation [4] . The staphylococcal enterotoxins are emetic toxins belonging to a large family of staphylococcal and streptococcal pyrogenic exotoxins (PT), having common structure, function phylogenetic relationships and sequence homology [10] .

Nowadays, there are 24 types of staphylococcal enterotoxins including staphylococcal enterotoxins (SEs) with demonstrated emetic activity in a primate model (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, SES and SET) and staphylococcal-like (SEl) proteins, which are not emetic in a primate model or have yet to be tested (SElJ, SElU, SElU2, SElV, SElX and SElY) [11] [12] , yet, SEA-SEE are the most common enterotoxins implicated in SFP [13] . SEA and SEB were found to be the most common staphylococcal enterotoxins. SEA is the most implicated toxin in staphylococcal food poisoning (SFP). For SEB, it has also been associated with food poisoning and was studied for the potential risk of being used as an inhaled bioweapon. SED is the second most common staphylococcal toxin associated with SFP in the world, and minute amounts of this toxin were found enough to induce illness. SEC was the most predominant enterotoxin detected in S. aureus strains isolated from mastitis milk in sheep, goats, and cattle. SEE has been reported in few cases of food poisoning [14] . SEG is an example of the non-classical enterotoxins which also show emetic properties [15] . The SEs are resistant to inactivation by gastrointestinal proteases such as pepsin and trypsin and these toxins also show thermal stability [10] making their elimination difficult to achieve [6] . Therefore, detection of SEs in foods is necessary for risk management and surveillance in food safety. Many reports have described the development of molecular methods for the detection of most of the SE genes [16] .

For classical staphylococcal enterotoxin detection, the reversed phase latex agglutination (SET-RPLA) and staphylococcal enterotoxin enzyme immuno-assay (SET-EIA) kits, were used to be employed. However, due to the unavailability of immunoassay kits for detecting new SEs, other methods as polymerase chain reaction (PCR) primers, DNA probes and reverse-transcription (RT)-PCR were used for the detection of these toxin genes and their activity in S. aureus strains [17] .

This study has been undertaken to detect enterotoxin genes in S. aureus by PCR, assess their distribution among various clinical and environmental sources and measure their expression levels by RT-PCR. Moreover, as the relationship between resistance and virulence is poorly understood, we tried to find any possible correlation between prevalence and expression of enterotoxins on one hand and antimicrobial susceptibility on the other hand.

S. aureus is a major human pathogen found in hospitals and the community [24] . This bacterium is an opportunistic pathogen that can result in a variety of self-limiting and also life-threatening diseases in humans [25] .

The clinical importance of S. aureus is attributed to remarkable virulence factors, toxins and enzymes, surface proteins, as well as the rapid development of antimicrobial agents resistance [26] . Among the most associated virulence factors with this bacterium, are the heat-stable staphylococcal enterotoxins (SEs) [27] . These toxins are the causative agents of staphylococcal food poisoning syndrome in man and they may be involved in other types of infections [28] .

Emergence of antibiotic resistant strains of S. aureus is a major public health problem since the bacteria can be easily spread in the environment. During the last two decades, the rate of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been notably increased world-wide [29] . Multidrug-resistant S. aureus have been frequently isolated from different origins, including food, water, human nasal mucosa and clinical cases [30] . Accordingly, it was extremely important in this study to detect the enterotoxigenicity as well as the antimicrobial susceptibility of S. aureus isolates belonging to various sources and try to find correlations between different variables.

In the present study, 100 S. aureus isolates were investigated for susceptibility against 12 antimicrobial agents belonging to four different classes (Figure 1). All of the isolates were resistant to ceftazidime. Also, 77% of isolates were resistant to amoxicillin/clavulanic acid. In a study of Manoharan et al. 2008, the percentage of resistance to this combination was 84.38% of isolates. These high levels of resistance may be attributed to misuse of antibiotics [31] .

Intermediate frequencies of resistance towards oxacillin, cefepime, ampicillin/sulbactam, erythromycin and tobramycin antibiotics (47%, 46%, 44%, 39% and 32% respectively) were recorded. The level of oxacillin resistance (47%) was higher than those reported by Sina et al. 2013 and Tokajian et al. 2011 (32%, 25%) respectively [32] [33] . Perveen et al. 2013 demonstrated that 12% of isolates were resistant to cefepime [34] which come in contrast with our finding. Opere et al. 2013 reported that erythromycin level of resistance was 32%, which was consistent with our results [35] . Resistance towards tobramycin in this study was coherent with Schmitz et al. 1999 who demonstrated that 29% of S. aureus isolates were resistant to tobramycin [36] , while Elazhari et al. 2011 showed that only 1.43% of isolates were resistant [37] .

For ciprofloxacin, 22% of isolates are resistant antimicrobial agent, Shittu and Lin, 2006 showed a lower level of resistance to ciprofloxacin (<10%) [38] , while Opere et al. 2013 showed higher level of ciprofloxacin resistance (39%) [35] .

The majority of our isolates were susceptible to amikacin and cephalothin (71%, 76%) respectively. This was also reported by Hauschild et al. 2008, Gade and Qazi, 2014 who demonstrated that 75.6% and 74% of S. aureus isolates were susceptible to amikacin respectively [39] [40] . Since cephalothin is a first generation cephalosporin so, it has good activity against S. aureus infections [41] . Imipenem antibiotic showed the highest activity towards isolates, where 87% of them were susceptible. This result was in accordance with the study of Asadollahi et al. 2014, in which 98% of isolates were susceptible [42] .

In this study, 88% of isolates were identified as MRSA depending on cefoxitin (30 µg) disk diffusion results [19] . Methicillin resistance was detected in S. aureus using cefoxitin with 100% specificity and sensitivity [43] . Also Alfatemi et al. 2014 demonstrated that cefoxitin had 100% sensitivity and 99% specificity for MRSA detection [20] . Our findings agree with the study of Yaseen et al. 2013, who reported that (88%) of isolates were MRSA [44] . Multidrug-resistance (MDR) was defined as resistance to at least 3 antimicrobial classes [45] [46] . 33% of our isolates were MDR, Seas et al. 2006 demonstrated a lower percentage, where 25% of isolates were MDR [45] .

The prevalence of five SEs encoding genes was investigated by PCR amplification. 88% of isolates harbored one or more enterotoxin genes. This result was to some extent in accordance with the studies of Elazhari et al. 2011 and Cao et al. 2012 who found toxigenicity levels of (76.4% and 93.5%) respectively [37] [47] , however, Becker et al. 2003 and Naffa et al. 2006, reported a lower level of toxigenicity 43% and 23%, respectively [48] [49] .

Among 88 SE genes positive isolates, only 16 different toxin patterns were detected (Table 2); this can be explained by the horizontal-gene transfer among strains [50] . Since all SE genes are carried on accessory genetic elements, including plasmids, genomic island vSa, S. aureus pathogenicity islands (SaPIs) and prophages, or next to the staphylococcal cassette chromosome (SCC) elements. Most of these are mobile genetic elements, which enable them to spread among S. aureus isolates increasing their ability to cause disease and contribute to the emergence of pathogenic strains [51] .

The sea gene was the most predominant toxin gene detected in 66% of isolates, this high percentage is in the line with other studies who reported sea gene prevalence rate of 55.1%, 65.22%, 60.6% [52] - [54] . However, a much lower frequency of sea gene was reported by other studies where sea gene prevalence was 25% and 18.86% [33] [55] . The second most prevalent toxin gene detected in this study was seb, where (38%) of isolates harbored this gene, this was in accordance with the study performed by Sina et al. 2013 who reported that 44.3% of isolates were seb gene carriers [32] . It was found that only one isolate (8%) out of twelve isolates from blood carried the seb gene, similarly only 5% of S. aureus isolates collected from blood stream infections harbored this gene [56] .

Regarding the sec gene, it was detected in 23% of isolates. This was in accordance with the results of Lim et al, 2012 who reported that the prevalence rate of sec gene was 19% [57] . However, higher prevalence rates for sec gene (61.3% and 41.5%) were reported [47] [55] . For see gene, only 19% of isolates harbored it. Likewise, see gene was harbored by 15.09% [55] . However, Asadollahi et al. 2014 reported a higher level of see gene (31%) [42] . In contrast, other studies did not detect see gene in any of the isolates [33] [47] [53] .

The prevalence of the non-classical (recently described) enterotoxin G encoding gene (seg) was also studied; it was the least prevalent gene in our study, where it was harbored by only 5% of isolates. Among S. aureus isolates collected from pediatric patients in Iran [55] , none of the isolates carried the seg gene. However, a higher level of seg gene was recorded in other studies, where the prevalence rate of this gene was 25% and 32.6% [33] [53] . The coexistence of two or more toxins was observed with a prevalence of 49%, higher combination rates (67.2%, 76.9%, 94.57%) were found in previous studies [47] [53] [58] .

The tested S. aureus isolates were distributed into 17 different patterns (T1-T17) according to their toxins. Patterns T1:T4 represents only a single gene, patterns (T5:T16) represent two or more toxin genes. Pattern 17 was for isolates devoid from toxin genes. The most common combinations in this study were (sea and seb) represented by pattern T5 (11%), followed by pattern T6 (sea and sec) genes (10%) and pattern T7 (sea and see) (9%). In a study of Kamarehei et al. 2013, 18.8% of isolates combined sea and seb genes [54] . Our data show that the coexistence of three toxin genes was found only in 8 isolates. A lower frequency (3%) was noticed for isolates harboring four toxin genes. However, some of our findings differed from those reported in other countries. In a study carried out in the Bronx, New York up to 12 SE genes were detected per a given S. aureus strain [59] . In our study, we did not observe any isolate harbored more than 4 enterotoxin genes.

Variations in enterotoxin encoding genes prevalence among studies may be attributed to differences in many factors, including the origin of the isolates (food, clinical or environmental samples), study locations, levels of hygiene restrictions among countries and also assay methods.

One of the most important finding in our study is that no statistically significant association between prevalence of toxin genes (seb, sec and see) and isolates sources, except for sea gene, which was significantly associated with clinical isolates (P value = 0.0059), where the environmental isolates did not show a remarkable increase in the toxin gene prevalence.

The relationship between virulence and resistance remains unclear. Some studies suggested a negative correlation between methicillin resistance and virulence, as resistance to antibiotics is associated with loss of pathogenic fitness and decreased virulence potential [60] [61] . Others observed that, in general, there were no differences between MRSA and MSSA in SEs levels [62] . In agreement with the study of Indrawattana et al. 2013 who found that sec gene was more associated with MSSA [53] . Our study confirmed that sec gene was significantly associated with isolates susceptible to cefoxitin (MSSA) (P value = 0.0281). Moreover, sea gene was more prevalent in isolates resistant to imipenem and ciprofloxacin antimicrobials with (P value = 0.0038 and 0.0237), respectively which was in accordance with Pourmand et al. 2009 who found a significant association between sea gene prevalence and resistance to a number of antimicrobial agents including ciprofloxacin [63] .

It is well known that detection of toxin genes, does not necessarily mean the ability of toxin production [58] , this may be due to mutation in the gene regulatory region [64] , so it was important for us to demonstrate the ability to express the studied enterotoxin genes among selected 20 isolates by real-time PCR technique. Sea gene was expressed in all the tested 20 isolates by variable degrees (Figure 2). In addition, seb gene was expressed in 11 out of 12 isolates that harbored this gene. However, only two of these eleven isolates showed relatively very high expression levels (Figure 3). For seg gene, it was expressed in all of the 3 tested isolates with two of them showing very high relative expression levels (Figure 4). Regarding sec and see genes, they were not expressed in any of the isolates tested by RT-PCR. The same results were obtained by Vasconcelos et al. 2011, where no expression of see gene was observed for any isolate [65] . Also, our finding was in agreement with Zigo et al. 2014, where sec gene was not produced in any of the tested isolates [28] .

Analysis of toxin pattern distribution among multidrug-resistant and non multidrug-resistant isolates did not allow the determination of a clear correlation between the determined toxin pattern distribution among MDR and NMDR. This is because 64.7% of patterns were shared by both MDR and NMDR. Our results were consistent with Indrawattana et al. 2013 who reported that no data was available in the literature correlating the ability to produce toxins in S. aureus and antibiograms [53] . However, an interesting correlation was found between high sea gene expression and multi-drug resistance (MDR) (P value = 0.0031). In addition, the 2 isolates that showed high seb gene expression as well as high seg gene expression were NMDR strains, but no significant correlation was found.

The present study highlights the high prevalence of enterotoxin genes, especially sea gene. The enterotoxin genes (seb, sec and see) were demonstrated to be strain dependent and not associated with the isolation source. However, sea gene was significantly associated with clinical isolates. Since detection of toxin genes does not necessarily mean the ability of toxin production, our results suggest that measurement of expression level of those toxins at the transcript level is necessary to evaluate the potential of S. aureus to cause severe disease. Although the genetic association between resistance and enterotoxins is poorly understood, evidence in our study indicates that there is a significant correlation between sea gene and both imipenem and ciprofloxacin resistance. Moreover, sec gene was significantly associated with MSSA isolates. An interesting correlation was also found between high sea gene expression and multidrug-resistance. Although isolates collected from hospitals environment did not show the expected antimicrobial resistance and toxin prevalence, more governed health restrictions are still needed to control the spread of S. aureus infections in our hospitals environment. Since these isolates may provide a source of fomite transmission, which may be associated with a high degree of antibiotic resistance and virulence.

All appreciation to Mansoura University hospitals staff members and patients for providing isolates used in the study. Many thanks for workers in Seeds Unit faculty of agriculture, Mansoura University.

The authors declare that they have no competing interests.

Shaymaa H. Abdel-Rhman, Rasha Barwa and Mohammed A. El-Sokkary conceived and designed the experiments; Enas Y. Mohammed performed the experiments; Shaymaa H. Abdel-Rhman, Rasha Barwa and Enas Y. Mohammed analyzed the data; Shaymaa H. Abdel-Rhman, Rasha Barwa and Enas Y. Mohammed contributed reagents/materials/analysis tools; all authors wrote the paper, read and approved the final manuscript.

Microbiology and Immunology Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.

Enas Yasser Mohammed,Shaymaa Hassan Abdel-Rhman,Rasha Barwa,Mohammed Adel El-Sokkary, (2016) Studies on Enterotoxins and Antimicrobial Resistance in Staphylococcus aureus Isolated from Various Sources. Advances in Microbiology,06,263-275. doi: 10.4236/aim.2016.64026