Studies on Enterotoxins and Antimicrobial Resistance in Staphylococcus aureus Isolated from Various Sources

Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins in S. aureus isolated from different sources and to evaluate the association of these toxins in comparison to susceptibility towards 12 antimicrobials; antimicrobial susceptibility was conducted by disc diffusion method. Detection of staphylococcal enterotoxins was performed by PCR and the ability to express these genes was assessed among isolates by RT-PCR. The most common enterotoxin gene was sea gene (66%), followed by seb, sec, see and seg (38%, 23%, 19% and 5%) respectively. Expression of sea, seb and seg genes was variable. However, sec and see genes were not expressed by any of the tested isolates. No statistically significant association exists between (seb, sec and see) and isolation sources, while the sea was significantly associated with clinical isolates. High significant correlation was found between elevated sea expression and multidrug-resistance. Our findings indicate that the pathogenic potential of S. aureus may be greater than previously thought. This emphasizes the utmost need to implement proactive measures and more emphasis will be placed on the application of hygiene practices in hospitals to control S. aureus infection and enterotoxins production.


Introduction
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.

Study Design
During the period from June 2013 and January 2014, 250 samples were collected from different hospitals.These isolates were obtained from different clinical and environmental sources.One hundred isolates were identified as S. aureus using standard microbiological techniques according to [18].The experimental protocol conducted in the study was approved by the Ethical Committee of faculty of Pharmacy, Mansoura University, Egypt, with code (2013-28) in May 2013.

PCR Detection of Staphylococcal Enterotoxins Genes
PCR amplification of staphylococcal enterotoxins (SE) genes, namely (sea, seb, sec, see and seg) was performed using a single plex PCR reaction (FPROGO2D, Tchne LTD, Oxford Cambridge, UK) and specific primers listed in Table 1.Genomic DNA was prepared by picking up one -two colonies grown overnight on nutrient agar medium and resuspended in 100 µl of nuclease free water then heated to 95˚C for 10 mins.The toxin genes were detected and amplified using the following reaction: 12.5 μl DreamTaq Green PCR Master Mix (2X) (Thermo scientific), 3 μl of bacterial DNA, 1 μl of each primer (10 µM), and 7.5 μl nuclease free water (Thermo scientific) for a total of 25 µl per reaction.
The PCR program started with an initial denaturation of DNA at 95C for 5 mins, followed by 35 cycles of denaturation at 95˚C for 30 s, annealing at temperatures specified for each primer as listed in Table 1 for 30 s and extension at 72˚C for 1 min.This was followed by a final extension step at 72˚C for 5 mins.The amplified genes were visualized on 2% agarose gel electrophoresis stained with ethidium bromide and compared with a 100 base pair (bp) DNA ladder (Thermo scientific).

RNA Isolation
RNA was isolated from 20 isolates.We selected isolates that harbored 3 or more of the tested toxin genes as detected by PCR (11 isolates), and isolates shared the same resistance and toxin gene pattern (9 isolates).RNA isolation was done according to [22] using glass beads and TRI reagent (Sigma Aldrich).
The concentration and the purity of RNA for each isolate were determined spectrophotometrically using 260 and 260/280 nm ratio respectively, using NanoDrop (ND-1000 Spectrophotometer, NanoDrop Technologies, Wilmington, Delaware, USA).The purity of RNA was determined by calculating the ratio of A260/A280.Pure RNA has a ratio of 1.8:2.1.

Expression of SE Genes
Isolated RNA was used for preparation of complementary DNA using Quanti-Tect Reverse Transcription kit (QIAGEN, Germany).RT-PCR was done using 5× FIREPol Eva Green, qPCR Mix, plus no ROX dye (Solis Bio-Dyne, Tartu, Estonia) using primers listed in Table 1.
RT-PCR was performed using a Rotor Gene Q thermocycler (QIAGEN, Hilden, Germany) programmed as follows: 95˚C for 15 min, followed by 35 cycles of denaturation at 95˚C for 15 s, annealing at temperatures specified for each primer as listed in Table 1 for 30 s and extension at 72˚C for 1 min.Expression of the target genes was normalized to the expression of reference gene (nuc) encoding nuclease enzyme.The gene expression level in samples was calculated relative to the housekeeping gene (endogenous reference gene) using a calibrator sample by the comparative (∆∆ct) method [23].All measurements were performed in triplicate.

Statistical Analysis
Correlations between data were statistically analyzed using the Graphpad Instat 3. Fisher's exact test was used to evaluate these correlations where P value < 0.05 was considered statistically significant.

Bacterial Isolates
A total of 250 samples were collected from different hospitals.One hundred were identified as S. aureus.These isolates were obtained from various sources (93 clinical isolates and 7 environmental isolates).

Antimicrobial Susceptibility Testing
As determined by disc-diffusion antimicrobial susceptibility testing, all isolates showed resistance to at least one antimicrobial agent.A high resistance percentage was observed for ceftazidime (100%), cefoxitin (88%) and amoxicillin-clavulanic (77%).A moderate activity was noticed for tobramycin, erythromycin, ampicillin-sulbactam, cefepime and oxacillin where (32% -47%) of isolates were resistant.On the other hand a lower resistance was observed for imipenem, ciprofloxacin, cephalothin and amikacin (Figure 1).
Among isolates, 88 were identified as methicillin resistant Staph.aureus (MRSA) where they showed resistance to cefoxitin.In addition, 33 were considered multidrug-resistant (MDR) (resistant to at least three different antimicrobial classes).

Polymerase Chain Reaction (PCR)
PCR analysis of the five toxin genes revealed that 88% of isolates harbored one or more of the tested    staphylococcal enterotoxin genes.The most frequent gene was sea (66% of isolates), followed by seb (38%), sec (23%), see (19%) and seg (5%).The tested S. aureus isolates were distributed into 17 different patterns (T1-T17) according to Table 2.The most predominant pattern was T1 involving 25 isolates harboring sea gene only.The second most common pattern was T5 representing 11 isolates harboring both sea and seb genes.However, patterns T12, T13, T14 and T16 were the least common, where only one isolate represented each pattern.
In addition, 39% of isolates harbored only one toxin gene (sea, seb, sec, see), while 49% carried toxin gene combinations (38% harbored 2 genes, 8% harbored 3 genes and only 3% harbored 4 genes. Among the 88 MRSA isolates, 76 harbored at least one enterotoxin gene and among them 56 isolates harbored the sea gene, 33 harbored seb gene, 17 harbored sec, 17 harbored see gene and seg gene was harbored by 4 isolates.The most common enterotoxin pattern in the MRSA toxigenic isolates was T1 coding for sea gene only (21 isolates) followed by T5 coding for sea and seb gene combination (11 isolates).

Real-Time PCR Analysis of S. aureus Enterotoxins
In order to study the relative expression of the tested genes, RNA was isolated from the selected 20 S. aureus isolates.The isolated RNA had an A260/A280 ratio that ranged between 1.766 and 2.12, while the concentation range was (0.352 -12.95) µg/µl.From each isolate 1 µg of RNA was used in c-DNA synthesis.The standard curve of the housekeeping gene nuc and of all expressed enterotoxins genes showed R 2 values of 0.97 -0.99.They showed the same melting profile and formation of pure amplicons indicated the specificity of the assay.The relative expression levels of SE were analyzed using the comparative method (2 −ΔΔCt ) method.
Among the tested twenty isolates, 20 harbored sea gene, 12 harbored seb gene, 12 harbored sec gene, 7 har-bored see gene and 3 harbored seg gene as detected by PCR.Sea gene expression was observed in all of the 20 isolates by variable degrees (Figure 2).Regarding seb gene, it was expressed in 11 out of 12 isolates (Figure 3).In contrast, sec and see genes were not expressed in any of the tested isolates, while the new (non-classical) enterotoxin gene (seg) was expressed in the 3 harboring isolates (Figure 4).
It was found that 70% of MDR isolates of the tested isolates expressed sea gene with high degree (P value = 0.0031) (Figure 2), seb and seg genes were highly expressed in 2 isolates that were non-multidrug-resistant (NMDR) and showed low sea gene expression (Figure 3, Figure 4) but no significant association was found.

Discussion
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].
The majority of our isolates were susceptible to amikacin and cephalothin (71%, 76%) respectively.This was also reported by Hauschild et al. 2008, Gade andQazi, 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].

Isolate code Non multi-drug resistant
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 andCao et al. 2012 who found toxigenicity levels of (76.4% and 93.5%) respectively [37] [47], however, Becker et al. 2003 andNaffa 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 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.

Conclusion
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.

Figure 2 .
Figure 2. Relative sea gene expression levels in 20 isolates calculated by the comparative method (∆∆ct) method.

Figure 3 .
Figure 3. Relative seb gene expression levels in 12 isolates calculated by the comparative method (∆∆ct) method.

Figure 4 .
Figure 4.The relative seg gene expression levels in 3 isolates calculated by the comparative method (∆∆ct) method. 8079

Table 1 .
Specific amplification primer sets of staphylococcal enterotoxin genes and reference gene (nuc).

Table 2 .
Distribution of enterotoxin gene patterns among isolation sources and resistance categories of S. aureus isolates.