Co-occurrence of sea, sec, and tst Enterotoxin Genes in Staphylococcus aureus isolates From Clinical Sources

1. Introduction
Staphylococcus aureus is a common pathogen that can inhabit various parts of the body and cause a variety of infections, such as skin and tissue infections, food poisoning, hospital-acquired infections, pneumonia, septic arthritis, endocarditis, osteomyelitis, foreign body infections, and sepsis [1, 2]. S. aureus has multiple virulence factors that contribute to its pathogenicity and bacterial colonization [3]. These virulence factors include drug resistance, enterotoxins, and toxic shock syndrome toxin (TSST). Enterotoxins and TSST are pyrogenic super antigens that react with the MHC II molecule, causing T lymphocytes to proliferate extensively and leading to damage through the release of high levels of cytokines. S. aureus has been identified as having more than 23 types of enterotoxins, which contribute to gastrointestinal poisoning and gastroenteritis [4-6]. The majority of S. aureus strains found in patients with toxic shock syndrome produce a harmful toxin called TSST-1, which can cause failure of vital organs and is often fatal [7-9]. In addition, antibiotic resistance is a significant problem in managing various hospital infections. It not only causes treatment failure in some cases but also increases hospitalization time and treatment costs [10-14]. Infections caused by S. aureus are becoming increasingly difficult to treat due to the widespread circulation and emergence of drug-resistant strains. Toxic shock syndrome is often treated with clindamycin and vancomycin. However, the excessive use and inappropriate prescription of antibiotics have led to an increase in antibiotic resistance, further complicating the treatment of toxic shock syndrome [7, 15]. Furthermore, there is a lack of research on human samples in Iran, as most of the existing studies have focused on food and animal sources. Therefore, further research is needed to investigate the frequency of the sea, sec, and tst genes in S. aureus isolated from clinical sources, to better understand the health risks that patients face.

2. Materials and methods

2.1. Bacterial samples and identification
The study was conducted in 2021 on 100 S. aureus isolates collected from different samples of patients and outpatients from Karaj City hospitals, including wound, blood, urine, sputum, nasal, and pharyngeal swabs. The samples were identified using specific culture media and various biochemical tests in a microbiology laboratory. All identified isolates were inoculated in nutrient broth containing 20% glycerol and stored at -20 °C for further experiments [16, 17].

2.2. Antibiotics susceptibility test
To determine antibiotic sensitivity, all isolates were tested using the agar disk diffusion method on Muller-Hinton agar medium. The testing was performed with 12 antibiotic disks obtained from Padtan Teb Co., including: oxacillin (1 µg), vancomycin (30 µg), cefoxitin (30 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), ciprofloxacin (5 µg), erythromycin (15 µg), clindamycin (2 µg), ceftazidime (30 µg), gentamicin (10 µg), tetracycline (30 µg), penicillin (10 U), and chloramphenicol (30 µg). The results were reported as sensitive, intermediate, and resistant based on the inhibitory zone. S. aureus strain ATCC 25923 was used as a positive control. Isolates were classified as multidrug-resistant (MDR) if they were resistant to at least one antibiotic from three different antibiotic families, based on the results of the antibiotic sensitivity test [18, 19].

2.3. DNA extraction
Initially, we isolated S. aureus strains and extracted DNA using the BetaPrep Genomic DNA Extraction Kit from BETA BAYERN, Germany. The quantity and quality of the extracted DNA were assessed using optical density (OD) 260/280 ratios and agarose gel (1.5%) electrophoresis. The extracted DNA was then preserved at -20 °C for future use.

2.4. Triplex PCR reaction
The triplex PCR reaction was used to confirm the presence of the analyzed genes in the studied isolates. Specific primers (Table 1) were used to determine if the isolates had the sea, sec, and tst genes. The reaction mixture contained a final volume of 30 µL, consisting of 15 µL of Amplicon’s Master Mix (which includes Maxer Mix 1X, Tris-HCl 0.5 M, MgCl2 2 mM, dNTPs 1.6 mM, Taq 0.04 Units/μL, and 0.5 µL of water), 1 µL (0.2 μM) of forward and reverse primer for each gene, 2 µL (20 ng) of template DNA, and 7 µL of double-distilled sterile distilled water. The genes were amplified using a thermal cycler (Applied Biosystem) under the following conditions: an initial denaturation at 96 °C for 5 minutes, followed by 35 cycles consisting of denaturation at 94 °C for 1 minute, annealing at 57 °C for 1 minute, and extension at 72 °C for 1 minute. After the final amplification, the temperature was kept at 72 °C for 10 minutes [20]. A negative control was used, where all reagents were used except for the template DNA. For the positive control, the standard strains of S. aureus ATTC 13565, ATTC 19095, and ATTC 25923 were used for sea, sec, and tst genes, respectively. The Triplex PCR reaction product was run on a 2% agarose gel with a 100 bp ladder and checked under ultraviolet light with a Gel document device [21, 22].

2.5. Statistical analyses 
After that, we analyzed the results using Microsoft Excel 2010 and SPSS software, version 16. We used Cramer’s V and chi-square test, and set the significance level at P≤0.05.

3. Results

3.1. Bacterial samples 
A total of 100 S. aureus isolates were collected from clinical sources in Karaj, with 49 from women and 51 from men. The average age of the patients was 46.51 years. The isolates were obtained from various sample types, with the highest percentage from blood (70%) and the lowest from wounds (2%). Other samples were taken from urine, sputum, nasal, and pharyngeal swabs. The frequency of S. aureus isolates in various clinical samples is presented in Figure 1. Our study found a significant relationship between the sample type and the presence of the sea gene in the S. aureus isolates (P<0.05).

3.2. Antibiotics susceptibility 
The following sentences refer to Table 2, which contains details of the results related to the pattern of resistance and sensitivity to antibiotics. The results of the disk agar diffusion method for antibiotic sensitivity testing showed that the 92(92%) isolates were resistant to penicillin, while 82(82%) isolates were resistant to ceftazidime. Additionally, 47(47%) isolates were resistant to tetracycline, 43(43%) to erythromycin, and 38(38%) to cefoxitin. Other antibiotics with less resistance included ciprofloxacin (36%), oxacillin (34%), and clindamycin (33%). Moreover, 23(23%) isolates were resistant to gentamicin, 16(16%) isolates to trimethoprim-sulfamethoxazole, and only four (4%) isolates to chloramphenicol. Notably, no vancomycin-resistant isolates were observed in the study. The results of the antibiotic sensitivity test showed that 48(48%) out of the total isolates were identified as MDR.

3.3. Presence of enterotoxin genes
Out of 100 isolates studied, 14 did not have the sea, sec, and tst genes, while the remaining 86(86%) isolates had at least one of these genes. Among the 86 isolates, 79% had the sea gene, 5% had the sec gene, and 43% had the tst gene. Moreover, 4% had both the sea and sec genes, 36% had the sea and tst genes, and 2% had both sec and tst genes (Figure 2). Only one (1%) isolate showed the presence of all three sea, sec, and tst enterotoxin genes. Statistical analysis revealed a significant correlation between the presence of the tst gene and MDR isolates (P<0.05). Moreover, the frequency of this gene was higher in MDR isolates. The gender of patients had a significant association with the presence of the sec and tst genes (P<0.05). The tst gene was more prevalent in female patients, while the sec gene was more prevalent in male patients.

4. Discussion
S. aureus is a significant pathogen for humans and has been a leading cause of both community-acquired and hospital-acquired infections for several decades. Despite antibiotic treatment, this microorganism frequently causes severe complications in hospitalized patients, and its increasing drug resistance has made treatment challenging. Genetically, this bacterium possesses genes that contribute to virulence, antibiotic resistance, and enterotoxin production, which can have dangerous effects on the host [7, 24]. Our study analyzed 100 S. aureus isolates and found that the highest resistance rate was observed for penicillin (92%), while the lowest resistance rate was observed for vancomycin (0.0%). The resistance pattern to other antibiotics was as follows: ceftazidime (82%), tetracycline (47%), erythromycin (43%), cefoxitin (38%), ciprofloxacin (36%), oxacillin (34%), clindamycin (33%), gentamicin (23%), and trimethoprim-sulfamethoxazole (16%). In this regards, Jafari-Sales et al. (2019) reported a penicillin resistance rate of 100% in S. aureus, which is consistent with the findings of another study [25]. In 2016, a study showed that no S. aureus isolates were resistant to vancomycin, which is consistent with the present study, but the highest percentage of antibiotic resistance was found for clindamycin, oxacillin, and trimethoprim-sulfamethoxazole [26] Reisi et al. (2014) reported that the highest percentage of antibiotic resistance was to penicillin and cefotaxime (100%), while the lowest was to vancomycin (0.5%), which is in accordance with our results [27]. Wu et al. (2023) also found that 100% of S. aureus isolates were resistant to penicillin, but no vancomycin-resistant isolates were found among the samples [28]. Another study found that the level of antibiotic resistance to penicillin among S. aureus isolates was 68.3%, which is similar to our results. However, this level of resistance was lower than what we reported [2]. In another study, 92.5% of S. aureus isolates were resistant to penicillin, and 10.5% were confirmed as vancomycin-intermediate S. aureus [29]. The results of these studies and our own demonstrate differences and similarities in the level of resistance to different antibiotics, which can be attributed to various factors such as geographical region and the type and number of collected samples. However, what is certain is the increasing rate of resistance in these bacteria.
The molecular results of the presence of enterotoxin genes in S. aureus isolates showed that 79%, 5%, and 43% of the isolates carried sea, sec, and tst genes, respectively. It was found that 36% of the isolates had both sea and tst genes, 4% had sea and sec genes, and 2% had sec and tst genes. Additionally, 1% of the isolates showed a positive presence of all three genes. In this regard, Goli et al. (2018) conducted a study on 49 S. aureus isolates, of which 34.7% were positive for the sea gene [30]. Various researchers from different parts of the world have reported different frequencies of the sea gene in S. aureus isolates. Some studies similar to our results, such as those conducted by Katayoon et al. (2017) [31] and Rahimi et al. (2014) [32], have reported a high frequency of the sea gene in S. aureus isolates, with 86.2% and 100% of the isolates carrying the gene, respectively. On the other hand, other studies, including those by Asgarpoor et al. 2018 [1] and Nashev et al. (2007) [33], have reported a lower level of the sea gene in S. aureus isolates, with carrier rates ranging from 16% to 47.4%. The frequency of the sec gene has been investigated in various studies, revealing a range of S. aureus isolates that harbor the gene. For instance, Goli et al. (2018) reported that 10% of S. aureus isolates carried the gene [30]. Other studies, such as those conducted by Eshraghi et al. (2009) [3], and Saadati et al. [34], reported frequencies of the sec gene in S. aureus isolates of 1.6%, and 9.5%, respectively. The frequency of the tst gene, which is one of the important toxins in the virulence of S. aureus, has been investigated in different studies. Mohammad Jani et al. (2018) reported that the prevalence of the tst gene in S. aureus isolates was 43%, which is in agreement with our results [35]. However, other studies have reported different frequencies of this gene in S. aureus isolates. For example, Ramazanzadeh et al. (2015) reported 81% [36], Parsonnet et al. (2008) reported 9% [37], and Becker et al. (2001) reported 18.2% [38]. After comparing the findings of various research studies with the outcomes of the current study, it is evident that some results are consistent while others vary. The discrepancies in the frequency of the genes analyzed can be attributed to diverse factors such as: geographical location, sample type, sample size, strain natural habitat, the overall health of the population being studied, the pattern of health behavior in clinical and community settings, and differences in the investigation methods and primers used in molecular studies.

5. Conclusion
Our study revealed high levels of enterotoxins and antibiotic resistance in S. aureus isolates, which can exacerbate hospital infections and contribute to the spread of antibiotic resistance. It is crucial to take action against the rise of S. aureus genes that produce enterotoxins and TSST in clinical sources. Treating infections caused by this bacterium can be challenging and lead to severe consequences. Therefore, prioritizing disease control and avoiding unnecessary antibiotic use is essential to prevent the spread of resistance.

Acknowledgements
The authors of this article express their gratitude to the following individuals and institutions for their assistance in conducting the study: the staff of the clinical laboratory of Imam Ali and Shahid Rajaei Hospitals, the Razi Laboratory of Karaj, and the knowledgeable experts of the Microbiology and Molecular Laboratory of the Microbiology Department at Islamic Azad University, Karaj Branch.

Compliance with ethical guidelines
The study was approved by the Research Ethics Committee of Karaj Branch, Islamic Azad University, Karaj, Iran (Code: IR.IAU.K.REC.1399.048). All ethical considerations were observed in the preparation of this manuscript.

Data availability
All data generated or analyzed during this study are included in the article.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
Conceptualization: Ebrahim Babapour and Reza Mirnejad; Methodology: Ebrahim Babapour; Investigation, formal analysis, and writing the original draft: Ameneh Sadat Sadeghi; Review and editing: Ebrahim Babapour, Reza Mirnejad and Majid Taati Moghadam; Project administration: Reza Mirnejad.

Conflict of interest
The authors declared no conflict of interest. 

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