Prevalence of enterotoxins B and C in clinical isolates of Staphylococcus aureus from Southwest of Iran

Introduction: Staphylococcus aureus enterotoxins play an important role in the incidence of food poisoning and various other syndromes are occurred such as staphylococcal scalded skin syndrome and toxic shock syndrome. Objectives: The purpose of the present study was to investigate the frequency of enterotoxin B and C genes among S. aureus isolates from clinical samples obtained from inpatients of a hospital by polymerase chain reaction (PCR). Materials and Methods: This cross-sectional study conducted during the period July to December 2015. Clinical samples including blood, urine, wounds and nasal mucosa samples were collected from a hospital in Ahvaz, southwest of Iran and screened for S. aureus strains by several phenotypic and biochemical tests. A total of 132 clinical samples were collected from which 60 samples were infected with the S. aureus strains. Then PCR assay was carried out to determine the entB and entC genes prevalence among these isolates. Results: Molecular analysis revealed that only 8 (13.3%) isolates harbored the entB and/or entC genes. Five strains (8.3%) contained only entB gene, 2 strains (3.3%) contained entC gene and only one strain (1.7%) was simultaneously positive for both entB and entC genes. There was no significant difference among various age groups regarding enterotoxin genes (P=0.551). Conclusion: These results demonstrated that enterotoxin-producing strains have a relatively low incidence in clinical samples in understudy hospital, but monitoring of their prevalence is necessary in regular screening programs in order to find the possible increase in their prevalence and prevention of their outcomes.


Introduction
Staphylococcus aureus strains play an important role in the incidence of clinical infections and food poisoning, as far as these microorganisms are introduced to the second or third cause of food poisoning (1).Virulence factors of S. aureus are exotoxins, surface proteins, extracellular enzymes and polysaccharide capsule (2).On the other hand, one of the most important pathogenic factors of this bacterium is enterotoxins family (3).Staphylococcal enterotoxins (SEs) are proteins with 26-29 kDa molecular weights that are heat-stable and resistant to pepsin digestion and thus can cause gastrointestinal disease and food poisoning (4).SEs and toxic shock syndrome toxin-1 (TSST-1) are super-antigens that mean they can activate a large population of T helper lymphocytes (CD 4 + ) leading remarkable cytokine secretion, immune system disorders and even death (5).Each of SEs do this by non-specific binding to a portion of class II

Key point
To investigate the frequency of enterotoxin B and C genes among S. aureus isolates from clinical samples obtained from in-patients of a hospital, we found that enterotoxin-producing strains have a relatively low incidence in clinical samples in a hospital of Ahvaz.However, monitoring of their prevalence is necessary in regular screening programs in order to find the possible increase in their prevalence and prevention of their outcomes.
MHC (major histocompatibility complex), for example staphylococcal enterotoxin B (SEB) bind to the alpha chain whereas enterotoxin E (SEE) bind to the beta chain of the class II MHC molecule (6,7).Today molecular methods, including polymerase chain reaction (PCR) and multiplex PCR as well as immunological techniques are used to identify enterotoxins-encoding S. aureus (8,9).Usually, enterotoxins-encoding genes are located on transposable genetic elements such as plasmids, prophages or DOI:10.15171/ipp.2018.24pathogenicity islands (10).Totally, enterotoxins family is consisted of 20 members that based on their biological function and serological characteristics are classified into two subfamilies of classic enterotoxins (staphylococcal enterotoxins A and E [SEA-SEE]) and new enterotoxins (staphylococcal enterotoxins G-U [SEG-SEU]) (11,12).It has been proved by many studies that not all of these enterotoxins cause food poisoning.SEA mostly causes S. aureus related-food poisoning compared to other enterotoxins (13).SEB while is related to food poisoning, is known as a bioterrorism agent (14).Additionally, SEC that is the third discovered enterotoxin, usually encoded by invasive S. aureus isolates, especially methicillin resistant S. aureus (MRSA) and is one of the main pollutants in milk that enable causes economic losses in the dairy industry (15,16).

Objectives
The aim of the present study is to investigate the frequency of enterotoxin B and C-encoding S. aureus isolated from clinical samples by PCR.

Materials and Methods
Clinical samples including blood, urine, wounds and nasal mucosa samples (5 mL for liquids and a sterile swab for wound and nasal mucosa) were collected during July to December 2015 from a hospital in Ahvaz, southwest of Iran.These samples were obtained from all patients referred to the pathology laboratory of the mentioned hospital.The bacterial isolates were identified based on several phenotypic and biochemical tests, including gram staining, catalase and coagulase analysis, growth on Mannitol salt agar (MSA) medium and susceptibility to furazolidone and resistance to bacitracin.In order to screening for the presence of entB and entC genes, PCR assay was performed.DNA was extracted by boiling a single colony of S. aureus was dissolved in 200 µL milli-Q water and heated at 100˚C for 15 minutes.Then the sample was centrifuged at 5000 rpm for 1 minute and the supernatant (containing DNA) was transferred to another Eppendorf tube.Cold ethanol 99% (Merck, Germany) was added to this supernatant (2.5 V/V) and stored at -22˚C for 1 hour.Following centrifugation at 13000 rpm for 10 minutes the supernatant was discarded and the sediment as template DNA was dissolved in 50 µL milli-Q water and stored at -22˚C.PCR reaction was carried out by specific primers for entB and entC genes (Table 1) in the 25 μL reaction mixture contained 12.5 μL of 2x PCR Master Mix Red (Ampliqon, Denmark), 10 ρmol of each primer, 1 μL of template DNA and 10 μL of milli-Q water.Concentration of MgCl 2 was 1.5 mM.The oligonucleotide primers for amplification entB and entC genes were selected based on the study of Mehrotra et al (17).PCR reaction was carried out by an initial denaturation step at 94°C for 5 minutes, 35 cycles each consisting of denaturation at 94°C for 2 min, annealing at 58°C for 2 minutes, and extension at 72°C for 1 minute followed by a final extension step at 72°C for 7 minutes in a thermal cycler (Bio-Rad USA).The amplified product was confirmed by electrophoresis in 1.5% agarose gel for 45 minutes at 80 V and documented by UVI gel doc system.

Ethical issues
The research followed the tenets of the Declaration of Helsinki.Consent for the study had been taken.The ethical committee of AJA University of Medical Sciences, Faculty of Medicine approved the research.

Statistical analysis
The obtained data were analyzed by SPSS software (version 22) through chi-square method with 95% confidence interval (P < 0.05).The t test and ANOVA tests were performed considering 95% CI and P value <0.05 as a significant result.

Results
In this research, 132 clinical samples were obtained from the pathology laboratory of a hospital that among them 60 (40.5%) S. aureus strains were detected after phenotypic and biochemical analysis.These samples were from blood (16, 26.7%), urine (22, 36.7%),wounds (8, 13.3%) and nasal mucosa (14, 23.3%).From these samples, 78.3% were from men and 21.7% were from women.Also maximum S. aureus infection was in the age range of 21 to 30 years (Table 2).The results of PCR assay indicated that 8 (13.3%) of isolates were harbored with entB and/ or entC genes among them 5 strains (8.3%) contained only entB gene, 2 strains (3.3%) contained only entC gene and only one strain (1.7%) were positive for both entB and entC genes.As depicted in Figure 1, strains of related by column 5 and 6 containing entB.From the total of 8 entB and entC-encoding S. aureus strains, maximum frequency belonged to urine by 4 strains (50%).Also, their number in men was more than women (5 to 3 strains).Related data by 8 entB and entC-encoding S. aureus strains are summarized in Table 3.

Discussion
As SEs play a major role in pathogenesis of this agent and determining of the prevalence of S. aureus strains encoding these toxins is of great importance in order to control of its food poisoning.Identification of these strains is based on both molecular and immunological methods (18,19).Advantage of molecular methods than immunological methods (such as ELISA and radioimmunoassay) is that in the first case, enterotoxin genes expression is not necessary and strains that produce toxin even at low levels can be easily detected (20,21).Thus, we used PCR assay for this purpose.
Staphylococcus aureus strains are widespread in hospitals and can be easily found on the surfaces and also internal space and can make a wide range of hospital infections including hospital acquired methicillin resistant S. aureus low frequency (5.6% and 7.5% respectively) ( 17) that confirms findings of present research.
In other study in 2011, detection of enterotoxin A, B, C and Q genes was carried out, which in line with our findings, entB and entC had nearly low prevalence by 15.8% and 9.5% respectively (29).In this research the abundance of entB is less than the entC that confirms our findings.Like many studies, current study shows that some isolated S. aureus strains have more than one enterotoxin gene (30).Also, using statistical analysis we have examined the relationship between entB and entC-encoding S. aureus frequency and clinical samples type, the patient's age and patient's gender.Due to being P value ≥ 0.05 in all three statistical analysis, thus no correlation detected between the enterotoxin-encoding strains frequency and the three factors mentioned.

Conclusion
These results demonstrated that enterotoxin-producing strains have a relatively low incidence in clinical samples in one hospital of Ahvaz, but monitoring of their prevalence is necessary in regular screening programs in order to find the possible increase in their prevalence and prevention of their outcomes.

Limitations of the study
This study is a single center study, which further investigations by larger samples are needed.

Authors' contribution
Study concept and design; FN.Acquisition of data; HM.Laboratory work and analysis and interpretation of data; SA and AG.

Conflicts of interest
There is no conflict of interest regarding the publication of this paper.

Ethical considerations
Ethical issues (including plagiarism, misconduct, data fabrication, falsification, double publication or submission, redundancy) have been completely observed by the authors.

Table 1 .
Nucleotide sequences, locations in genes and sizes of PCR products for entB & entC genes-specific primers (16)reviously mentioned, the majority of S. aureus isolates in dairy products encodes high level of SEC(16).Therefore by high presence SEC in a clinical sample, it seems that people have used the contaminated milk and this shows the importance of studying the enterotoxin C. The results of current study revealed that from 60 detected S. aureus strains just 8 strains (13.3%) possessed enterotoxin B and C -producing genes.In addition, the prevalence of entB and entC is usually low in other research on enterotoxin genes frequency, while entA has usually high prevalence in among all the enterotoxins (27).

Table 2 .
Number and frequency (%) of clinical samples infected to Staphylococcus aureus based on age range