Prevalence of enterotoxin genes (SEA to SEE) and antibacterial resistant pattern of Staphylococcus aureus isolated from clinical specimens in Assiut city of Egypt

Infections in communities and hospitals are mostly caused by Staphylococcus aureus strains. This study aimed to determine the prevalence of five genes (SEA, SEB, SEC, SED and SEE) encoding staphylococcal enterotoxins in S. aureus isolates from various clinical specimens, as well as to assess the relationship of these isolates with antibiotic susceptibility. Traditional PCR was used to detect enterotoxin genes, and the ability of isolates expressing these genes was determined using Q.RT-PCR. Overall; 61.3% (n = 46) of the samples were positive for S. aureus out of 75 clinical specimens, including urine, abscess, wounds, and nasal swabs. The prevalence of antibiotic resistance showed S. aureus isolates were resistant to Nalidixic acid, Ampicillin and Amoxicillin (100%), Cefuroxime (94%), Ceftriaxone (89%), Ciprofloxacin (87%), Erythromycin and Ceftaxime (85%), Cephalexin and Clarithromycin (83%), Cefaclor (81%), Gentamicin (74%), Ofloxacin (72%), Chloramphenicol(59%), Amoxicillin/Clavulanic acid (54%), while all isolates sensitive to Imipinem (100%). By employing specific PCR, about 39.1% of isolates were harbored enterotoxin genes, enterotoxin A was the most predominant toxin in 32.6% of isolates, enterotoxin B with 4.3% of isolates and enterotoxin A and B were detected jointly in 2.1% of isolates, while enterotoxin C, D and E weren’t detected in any isolate. This study revealed a high prevalence of S. aureus among clinical specimens. The isolates were also multidrug resistant to several tested antibiotics. Enterotoxin A was the most prevalent gene among isolates. The presence of antibiotic resistance and enterotoxin genes may facilitate the spread of S. aureus strains and pose a potential threat to public health.


Background
Staphylococcus aureus is a commensal and opportunistic human pathogen that can be found all over the world [1]. It causes a variety of clinical infections, including impetigo, furunculosis, and abscesses on the skin and soft tissues, as well as systemic infections, including pneumonia, bacteremia, endocarditis, and toxin-mediated diseases [2]. It is one of the most common pathogens linked to nosocomial infections in hospitals [3]. S. aureus cause a large amount of morbidity and mortality in developing countries as opposed to other infectious diseases like malaria, tuberculosis, and HIV infections [4]. The fast expansion of drug resistance as well as prominent virulence factors, surface proteins, metabolites and enzymes have all contributed to S. aureus clinical significance [5]. Large numbers of toxins, including hemolysin (α, β, γ, δ) and leukocidin (PVL, Luk E/D) are the most commonly associated virulence factors with these microorganisms [6]. Other virulence factors that cause enterocolitis, scalded skin syndrome, and toxic shock include heat-stable staphylococcal enterotoxins (SEs), exfoliative toxins (ETA and ETB), the toxin of toxic shock syndrome-1(TSST-1) [7,8]. S. aureus is a concern not only because of its widespread distribution and pathogenicity but also because of its ability to resist antimicrobials [9]. In addition to drug resistance, monitoring of S. aureus strains and determination of susceptibility patterns are critical [10]. Staphylococcal enterotoxins have been identified as etiologic agents of human food poisoning and as active immunologic superantigens that induce non-specific T cell proliferation [11]. Enterotoxins are emetic toxins linked to a broad family of pyrogenic exotoxins produced by staphylococci and streptococci, they are active in concentrations ranging from nanograms to micrograms and are resistant to heat and low pH, also have proteolytic enzymes allowing them to remain active in the digestive tract after ingestion [7]. There are 23 staphylococcal superantigens that have been described, enterotoxins A to E, G to J, R to T, and staphylococcal enterotoxin-like toxins (SE1) K, Q, U, and X (SElK-SElQ, SE1U-SElX) [12][13][14][15][16][17]. Polymerase chain reaction (PCR), DNA probes, and reverse-transcription RT-PCR were used to detect enterotoxins and their activity in S. aureus strains [18]. So the aim of this research is to use PCR to detect enterotoxins genes in S. aureus, assess their distribution through clinical sources, and quantify their prevalence. Furthermore, we looked for any possible connection between enterotoxin prevalence and expression on the one side and antimicrobial susceptibility on the other.

Collection of samples
The present descriptive study was carried out over a six-month period, between January and July 2015, the research was approved by the ethics committee of the Botany and Microbiology department-the College of Science (Al-Azhar University, Assiut). A total of 75 clinical specimens were collected under aseptic conditions from patients admitted to Assiut University hospital-Faculty of Medicine, Political hospital, Al-Eman hospital, and El-Shmla hospital in Assiut governorate, Egypt. All clinical specimens collected from diverse infections were received from both genders and all age groups. Urine from the infected urinary tract (n = 29; 38.6%), Pus swabs from an abscess (n = 20; 26.6%), Swabs from septic wounds (n = 15; 20%), and Nasal swabs from cases with respiratory symptoms (n = 11; 14.6%). The samples obtained from patients were transported directly to the laboratory in an icebox within 4 h of collection and were immediately processed according to standard microbiological procedures. The sampling swabs were inoculated into a 5 ml Staphylococcus enrichment broth medium [19], blended briefly as necessary, and incubated at 37 °C for 24 h.

Prevalence and characterization of S. aureus isolates
The bacterial culture was streaked on mannitol salt agar (MSA) (Oxoid, Basingstoke, Hampshire, England), with a loopful of enrichment broth and incubated aerobically at 37 °C for 24 to 48 h. Suspected colonies were picked up, sub-cultured once on MSA and twice on 5% defibrinated sheep blood agar medium (Oxoid, Basingstoke, Hampshire, England). Bacterial colonies showing typical characteristics of S. aureus (i.e., beta-hemolytic on blood agar and colonies with golden yellow pigmentation on MSA) were subjected to subculture on to basic media, Gram stain, and biochemical tests catalase and coagulase. Catalase positive and Gram-positive bacteria appearing in the grape-like cluster was spot inoculated to DNase agar (Oxoid, Basingstoke, Hampshire, England). Inoculated DNase agar plates were incubated at 37 °C overnight and flooded with 1 N HCl (Merk, Darmstadt, Germany). Isolates that hydrolyzed DNA in DNase agar were considered S. aureus. Purified colonies were selected, propagated on nutrient agar slope, and preserved at 4 °C.

Antibiotics susceptibility testing
The disc diffusion method was used to determine the antibiotic susceptibility of S. aureus isolates on Mueller Hinton Agar (Oxoid, Basingstoke, Hampshire, England) [20]. Sixteen antibiotics were tested, Imipinem

Real-Time Q-PCR data analysis
The relative expression ratio was quantified and calculated accurately. Accordingly, for each biological sample, the difference (Δ) in quantification cycle value (C T ) between the target (C T (target) averaged from three technical repeats) and the reference 16S rRNA was used as the reference gene (C T (reference) , a fixed C T value was used for all samples). The C T (threshold of the cycle) value of each detected gene was determined by automated threshold analysis on ABI System [26]. The C T value of each target gene was normalized to C T (reference) to obtain ΔC T (target) where The relative expression quantity of the target gene was indicated with

Sequencing, sequence analysis, and phylogenetic tree construction of highly expression isolates
Amplified products of (16S rRNA gene and specific enterotoxin genes) for high expression isolates were purified according to (Maxim biotech INC, USA) and subjected to DNA sequencing using a forward primer in the sequencing reaction [27]. Sequencing was performed using BigDye ® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and model 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained DNA nucleotide sequences were analyzed using NCBI-BLAST (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) for confirming the identity of the obtained sequences. Multiple sequence alignment of our sequences and the other published ones were performed using CLUSTLAW (1.83) [28] and a phylogenetic tree was analyzed and generated using MEGA 4 [29].

Statistical analysis
Correlations between data of antibiotic resistance and prevalence of enterotoxins were statistically analyzed using the Graphpad Instat 3. Fisher's exact test was used to evaluate these correlations, where P value less than 0.05 was considered statistically significant.

Isolation and identification of S. aureus isolates
In total, 46 S. aureus isolates were isolated from 75 patient samples (29 urine samples, 20 abscess samples, 15 wound samples, and 11 nasal swabs samples). Suspected colonies turned MSA medium yellow due to mannitol fermentation and were given a clear zone around colonies on blood agar because of β-hemolytic activity of isolates. S. aureus isolates were found most often in abscess swabs (15 isolates from 20 samples; 75%) and then wound swabs (9 isolates from 15 samples; 60%), urine samples (16 isolates from 29 samples; 55.1%), and lastly nasal swabs (6 isolates from 11 samples; 54.5%) ( Table 2). Data are presented in Table 3 concluded that S. aureus isolates were positive for catalase, coagulase and, DNase, growth on crystal violet agar were purple, white and yellow colonies, also growth on Baird Parker agar medium were black and shiny with narrow white margins surrounded by clear zone, it also positive for Nitrate reduction, Methyl red test, Voges Prausker test and their ability for hydrolysis of Gelatin and Casein while negative for Indole production and Citrate utilization.

Susceptibility of S. aureus isolates to antibiotics
The disc diffusion method was used to screen all of the bacterial isolates on Muller-Hinton agar for sixteen antibiotics. The results revealed that all isolates were sensitive to imipinem (100%), 46% of isolates sensitive to amoxicillin/clavulanic acid, 41% sensitive to chloramphenicol, 28% sensitive to ofloxacin, 26% sensitive to gentamicin, 19% sensitive to Cefaclor, 17% sensitive to cephalexin and clarithromycin, 15% sensitive to erythromycin and ceftaxime, 13% sensitive to ciprofloxacin, 11% sensitive to ceftriaxone, 6% sensitive to cefuroxime, while  current results demonstrated all isolates resistant to nalidixic acid, ampicillin, and amoxicillin (Fig. 1).

PCR amplification of enterotoxin genes
The prevalence of different five enterotoxin genes in S. aureus isolates was investigated using PCR, it was reported that 15 isolates (32.6%) were positive for the SEA gene, 2 isolates (4.3%) were positive for the SEB gene, and only one isolate (2.1%) was positive for both the SEA and SEB genes (Fig. 2), SEC, SED and SEE genes were not found in any of the tested isolates.

Real-time PCR data analysis of S. aureus enterotoxins
The relative gene expression was examined of the tested harbored genes by isolation of RNA from the selected S. aureus isolates and gene expression was verified using a Reverse Transcription Real-time PCR, which enabled comparison of the target and reference genes, where the 16S rRNA was used as the reference gene and the   Fig. 3). The current report found that MDR of the tested isolates expressed SEA gene with (P value = 0.01) and SEB gene was expressed with (P value = 0.03).

Identification of highly expression isolates and phylogenetic analysis
Completely identification of S. aureus isolates exhibited high level of enterotoxin A and enterotoxin B expression; 16S rRNA gene and enterotoxin genes were amplified and sequenced. The annotated sequences were deposited in GenBank under accession number MF563554 Azhar1 strain for enterotoxin A producing strain and MF563555Azhar2 strain for enterotoxin B producing strain. Sequence alignment and phylogenetic tree analysis revealed that MF563554 given the similarity of about 96% with LT677428 and LT677437 human strains isolated from the head and neck tissue in the USA, while MF563555 had similar of about 90% with HM452073 isolated from bovine mastitis in India (Fig. 4). High expression isolates for SEA and SEB genes using specific primers were amplified and sequenced. The annotated sequence was deposited in GenBank under accession number LC315607 ELBAZ1 for enterotoxin A strain and MF621929 ELBAZ2 for the enterotoxin B strain. Sequence analysis revealed that LC315607 ELBAZ1 strain given 98% similarity with KX777250 which was a local isolate in Egypt while MF621929 ELBAZ2  strain given similar 99% with AB860415 strain isolated from Tokyo food poisoning outbreak and AB716349 isolated from a human nasal swab in Japan. The relationship between ELBAZ1 and ELBAZ2 among other standard strains available in GeneBank were assessed by constructing a neighboring-Joining tree (Figs. 5, 6).

Discussion
S. aureus is a widespread human pathogen that can be found in both hospitals and the public, it's an opportunistic pathogen that can cause several diseases in humans, both self-limiting and life-threatening [30,31]  (61.3%) of isolates. This is higher isolation rate than seen in earlier research (28.1% and 24.5%, respectively) [31,32]. The higher isolation of S. aureus was observed especially in children and neonates and this finding consistent with report [31], it is believed that their immunity is not properly developed at this phase to cope with bacterial illness hence they are vulnerable and easily infected especially when hospitalized. The older children have also been observed to be more active than adults during their interaction with their playmates and while playing for hours, come in contact with various objects. In this process, they become a target to ubiquitous bacteria such as S. aureus. On MSA and blood agar media, purified S. aureus colonies were smooth, circular, convex, entire, and given different pigments: yellow (47.8%), white (47.8%), and lemony yellow (4.3%), the present data supported through other investigations [32][33][34]. S. aureus isolates were found to have the highest occurrence rate in abscess specimens, which is consistent with previous results [32,33,35]. In contrast, the highest incidence rate of S. aureus was showed in urine specimens [36], wounds infections [37,38] and nasal swabs specimens [39,40]. The high occurrence of S. aureus was found in abscess specimens which could be attributed to poor personal hygiene and abscess exposure, making it more susceptible to contamination and infection. In addition, some people in the study region treat their abscesses with self-medication or by employment unqualified or poorly trained quacks before seeking proper medical attention, which might account for the level of settlement by S. aureus. Regarding antibiotic resistance, the current results were explained, all S. aureus isolates were resistant to ampicillin, amoxicillin, and nalidixic acid, these findings are consistent with those published in other studies [41,42]; this suggests that these antibiotics are no longer successful against infections caused by S. aureus. The other studied antibiotics showed a wide range of resistance like Cefuroxime (94%), Ceftriaxone (89%), Ciprofloxacin (87%), Ceftaxime and Erythromycin (85%), Cephalexin and Clarithromycin (83%), Cefaclor (81%), Gentamicin (74%), Ofloxacin (72%), Chloramphenicol (59%). and Amoxicillin/Clavulanic acid (54%), these data were compatible with some reports [32,43,44]. The present data were revealed significantly higher resistance to the tested antibiotics than other studies [33,34,45]. As a result of the high prevalence of antibiotic resistance in strains, antibiotics commonly used to treat S. aureus infections may not be sufficient. So, physicians must take into account the care guidelines for MRSA infections. All isolates, on the other hand, were found to be susceptible to Imipinem; this finding was consistent with prior investigations [46,47] that found 87% and 98% of isolates to be susceptible to Imipinem, respectively. So this medication is still successful and can be used as an additional treatment choice for S. aureus infections in the study area. It's possible that the great prevalence of resistance to the antibiotics mentioned is attributable to their widespread usage in the treatment of human diseases. This suggests that these antibiotics are no longer effective as an empirical treatment for S. aureus infections in the research field. The low activity of these antibiotics could be related to earlier exposure to these drugs that would have accelerated the development of resistance. The rise in antibiotic abuse in our region, which stems from self-medication, failure to react to care, and antibiotic-sale actions, can bolster this assertion. A long history of effective SE determination in epidemiology is cited in both clinical and environmental settings [48]. Enterotoxin genes were discovered in 39.1% of the isolates. Some investigations [49][50][51] found lower levels of toxigenicity (36%; 43%, and 23%, respectively). However, several studies revealed greater levels of enterotoxigenicity (88%, 76.4%, and 93.5%, respectively) [46,52,53]. Only two distinct enterotoxin genes were revealed among five enterotoxin genes, which was consistent with the findings of a previous investigation [50]. The enterotoxin A gene was found in 32.6% of isolates, which was similar to other studies [51,[54][55][56]  The SEB gene was found in 21.6% of bovine mastitis cases [63] and 24% of cutaneous infections [64]. SEA and SEB genes were discovered together in only one isolate (2.1%), which is lower than studies [46,65], which detected SEA and SEB genes combined in 11% and 22% of isolates, respectively. SEC, SED, and SEE genes were not found in any of the examined isolates; nevertheless, additional findings revealed the existence of the SEC, SED, and SEE genes in isolates [46,51,56,66]. It is well recognized that the presence of toxin genes does not imply the potential to produce toxin [46,67]. As a result, we used the real-time PCR technique to demonstrate the ability of chosen isolates to express the examined enterotoxin genes. With 100% and 80.66%, respectively, two isolates (10 and 42) showed extraordinarily high levels of expression. Furthermore, the SEB gene expression levels were demonstrated in varied degrees in isolates (5, 8, and 11) with a percentage ranging from 66 to 100%. These findings revealed considerable heterogeneity in the expression of enterotoxins among isolates, which was consistent with prior findings [46]. The discrepancies in the prevalence of enterotoxin encoding genes between studies can be due to a variety of factors, including the origin of the isolates, research locations, hygiene restrictions in different countries, and assay methods. The correlation between enterotoxin and resistance to antibiotics is unknown, but the current study demonstrated a strong connection between the determined toxin pattern distribution and antibiotic resistance and that was statically significant with (P value = 0.01 and > 0.05) for SEA and SEB genes respectively, and this was consistent with prior investigations where more association between level of enterotoxins and resistance to antibiotics [46,68,69], although some studies suggested a negative correlation between antibiotic resistance and enterotoxin investigation [70,71]. Also, one of the most important findings in the current investigation was the pigmentation of isolates; although, the relationship between pigment creation on MSA and enterotoxigencity remains unclear; however, there was a putative link between isolate pigmentation and enterotoxin generation. These findings corroborated those investigations that noticed that SEs genes were more correlated with antibiotic resistance and pigment development [69,70].

Conclusion
The current research looks at the prevalence of enterotoxin genes in clinical samples, especially the SEA gene, which is followed by the SEB gene. The source of isolation was not related to the enterotoxin genes. On the other hand, the SEA gene was discovered to be closely linked to clinical isolates. The existence of toxin genes does not necessarily mean that the toxin can be produced. Our results suggest that assessing S. aureus potential to cause serious disease requires evaluating the degree of expression of certain toxins at mRNA level. Despite the fact that the genetic relationship between resistance and enterotoxins is poorly understood, evidence from our study indicates that enterotoxin and antibiotic resistance are linked in a significant way.