Streptococcal Pyrogenic Exotoxin Genes SpeA and SpeB in Isolates of Streptococcus pyogenes from Children with Pharyngitis, Gezira State, Sudan

Background: Streptococcus pyogenes (group A streptococcus, GAS) is an important human bacterial pathogen. This organism possesses many virulence factors, Streptococcal pyrogenic exotoxinone of these. Aim: Detection of Streptococcal pyrogenic exotoxin SpeA and SpeB in isolated Streptococcus pyogenes. Methods: Tow hundred throat swab samples were collected from children with pharyngitis referred to Pediatric Teaching hospital and ENT hospital Wad medani, Sudan, from January to November 2021. The ques-tionnaire was filled out to collect clinical and demographic data. Throat swabs were collected and processed with the standard microbiological pro-cedure to isolate Streptococcus pyogenes. Antimicrobial susceptibility testing was done on all GAS isolates using the Kirby Bauer disk diffusion method according to clinical laboratory standard institute (CLSI) guidelines. Detection of Spy 1258 gene and Streptococcal pyrogenic exotoxins SpeA and SpeB were done by using Multiplex PCR. Results: Amongst the Tow hundred collected samples fifty-one isolates (25.5%) were identified as S. pyogenes. Antibiotic susceptibility testing showed that all the GAS isolates were sensitive to Azithromycin and Penicillin. Sensitivity to Erythromycin, Gentamicin, Clarithromycin, Amoxicillin and Cephalexin were 88.2%, 86.3%, 45.1%, 41.2%, 13.7%, respectively. SpeA was detected in 17 (33.3%) and SpeB in 48 (94.1%). Conclusion: Streptococcal pyrogenic exotoxin genes SpeA and SpeB were detected in 17 (33.3%) and 48 (94.1%) respectively of Streptococcus pyogenes isolates.


Introduction
Streptococcus pyogenes (Group A Streptococci GAS) cause a diverse of human diseases, from uncomplicated superficial infections of the respiratory tract and skin to severe invasive diseases associated with high morbidity and mortality.
Many virulence factors of GAS have been identified, including bacterial surface proteins, secreted streptolysins, hyaluronic acid capsule hyaluronidase, streptokinase and DNase [1], and among the major virulence factors of GAS are the secreted Streptococcal pyrogenic exotoxins (Spe), which act as superantigens (SAg) due to their ability to interact with the host major histocompatibility complex (MHC) class II molecules and with the changeable region of the T-cell receptor β-chain without previous processing by antigen-presenting cells. This interaction ends up in the activation of an oversized range of T-cells. Eleven distinct SAgs are known in GAS all of them sharing a standard macromolecule fold and therefore, the same target receptors on host cells: 3 chromosomally encoded (SpeG, SpeJ, and SMEZ) and eight encoded on temperate phages (Spe A, Spe C, Spe H, Spe I, Spe K, Spe L, Spe M, and SSA). Spe B and Spe F are each encoded on the microorganism body. Multiple forms are recognized in Spe A, Spe C, Spe G, SSA, and SmeZ that will be related to variations in super-antigenic activity and in substance properties [2]. The severity of GAS infections depends on multiple host and microorganism factors. The infective properties of GAS strains square measure typically coupled to the assembly of virulence factors like toxins, proteases or DNases and toxins gifted within the explicit GAS strain are often the predictor of its invasiveness [3].
The results from many studies recommend that the genetic background of the host may play a vital role in invasive illness conditions [4]. Superantigens contribute to GAS pathogenicity supported by their immune stimulatory activity [5]. Superantigens distribution has been used as a technique for the detection of genomic heterogeneousness, the correlation between gene contents and therefore, the determination of clinical manifestation [6]. Their biological toxicity and environmental stability have resulted in some superantigens being classified as chosen agents of the terrorist act [7]. The Spe toxins measure contributes to the pathologic process of severe invasive unwellness and has additionally been involved in reaction disorders [8]. The streptococcal pyrogenic toxin B (SpeB), is the predominantly secreted cysteine protease of GAS acid enzyme of GAS. SpeB

Methods
A cross-sectional study was done and samples were collected from children attending Pediatric Teaching Hospital and (ENT) hospital Wad medani, Sudan with symptoms of Pharyngitis, ages 5 to 17 years from January to November 2021. Exclusion criteria included Children with respiratory tract symptoms such as rhinorrhea or nasal congestion and prior antibiotic therapy in less than 7 days.

Ethical Approval
The study was approved by the Ethics Committee of the Ministry of Health, Gezira State.

Isolation of Bacteria
Throat swabs were inoculated on 5% sheep blood agar plates and incubated in 5% -10% CO 2 at 37˚C for 24 h. Identification of GAS isolates was made based on beta-hemolytic activity on sheep blood agar, small colony characteristics, Gram stain positive cocci (Streptococci), catalase production negative, 0.04-U bacitracin disc susceptiblility and PYR test were positive.

PCR Amplification of SpeA and SpeB
Streptococcal pyrogenic exotoxin genes were detected by using the Multiplex PCR amplification method. The target genes included Streptococcal Pyrogenic exotoxin genes SpeA and SpeB. Spy 1258 was used as an internal control.

DNA Extraction
DNA was isolated from Streptococcus pyogenes using standard laboratory protocol. DNA was extracted by Boiling method, S. pyogenes isolates were placed in 500 μL of Trise EDTA (TE) buffer and frozen in −70˚C, after thawing 150 μL of this suspension was taken and heated at 95˚C for 30 minute, centrifuge was used and spin at 14,000 rpm for 5 minute at 25˚C and the supernatant was taken into a new sterilized tube.

Primer Design
Identification of SpeA and SpeB genes by Polymerase Chain Reaction (PCR) was done by using primers designed according to Table 1.

PCR Amplification
Amplification reactions were performed for 35 cycles using PCR program shown in Table 2.

Gel Electrophoresis
Amplicons were visualized on 1.5% Agarose gel by Electrophoresis; the PCR products were electrophoresed through agarose gel with current 120 V for about 30 min. Gels are photographed under UV light.

Statistical Analysis
Statistical analysis was done by SPSS statistical software. Participants' demographic and clinical characteristics were described by using descriptive statistics.

Results
In this study, a total of 200 throat swabs were collected from Pharyngitis children from January to November 2021. Females accounted 126 (63%) and males were 74 (37%). The rate of Streptococcus pyogenes was 25.5% (51/200) which has been identified by using culture and biochemical tests. Total number of males infected by S. pyogenes was 13 (25.5%) and females were 38 (74.5%).
Resistance of Cephalexin was 70.6%; there was no significant association between the patients having SpeA and SpeB genes and resistance of bacteria to Cephalexin in Table 3 and Table 4.
There was a significant association with P-value between the patients having SpeA gene and the presence of cervical lymphadenopathy (Table 6). And there    (Table 7) more than patients who haven't this gene.

Discussion
The outcome of GAS infection is not only strain related but is also related to a combination of several factors, such as exotoxin production and host immunity.
Bacterial characteristics may also play a pathogenic and important role in the severity of streptococcal infections [9].
Streptococcus pyogenes is sensitive to penicillin; different studies worldwide showed that like [10] study from Iran, [11] study from Senegal, [12] study from Ethiopia, [13] study from China and our study also showed that which confirms the penicillin is still the drug of choice for the treatment of GAS pharyngitis.
Resistance to antimicrobial to Streptococcus pyogenes is variable and the emergence of drug resistance among streptococci to macrolides (erythromycin  [14]. Our study showed the resistance to Clarithromycin was 33.3% similar to [10], showing resistance 33.9% and the resistance to Erythromycin was 7.8% agree with 9.7% of [15] study and 6.9% of [9] study. And also show 52.9% resistance to amoxicillin, whereas the study of [5] from Egypt showed 81% sensitivity to amoxicillin.
The Superantigen SpeA was found in 33.3% of this study and SpeA was 36.8% in the study of [5]. Also, this result is similar to [13] study which showed the percentage of SpeA gene was 34.34%. A study of [9] showed a lower percentage of SpeA (17.2%) SpeB gene was detected in 94.1% of Streptococcus pyogenes isolates and showed 100% specificity agrees with [16] study that showed 100% sensitivity and 100% specificity. Among the toxin genes that are thought to be chromosomally encoded, SpeB was found in all isolates [17]. This finding differs markedly from that reported in [9] which showed 72.4% of SpeB. And up-close to [17] showed the presence of SpeB was 100% in their isolated Streptococcus pyogenes, also reporting the SAg and antibiotic resistance genes appeared to be associated with the emm type. The streptococcal pyrogenic exotoxin B gene was associated with pyrogenicity, T-lymphocyte mitogenicity, and the ability to increase susceptibility to endotoxic shock in individuals infected with group A streptococcus [18].
Our study showed there was a significant association between the patients having SpeB gene and fever. SpeA was detected in 33.3% of streptococcus pyogenes isolates. [4] Reported the SpeA gene was found in a majority (40% -90%) of S. pyogenes isolates from the USA associated with invasive disease and STSS, but only in a minority (15% -20%) of isolates from noninvasive diseases. A high frequency of SpeA (80%) was found in STSS isolates collected in Australia. In [9] study isolates from patients with pharyngotonsillitis, the frequencies were 17.2% for SpeA, 72.4% for SpeB. And in [19] the incidence of SpeA, SpeB and SpeF were 5 (83%), 56 (933%) and 53 (883%), respectively.