Introduction

Although GAS (especially S. pyogenes) has been in existence for many years, it remains as an important cause of global morbidity and mortality in resource-limited regions. S. pyogenes, which is a significant human pathogen, causes serious diseases, such as pharyngitis, skin infections, acute rheumatic fever (ARF), rheumatic heart disease (RHD), acute post-streptococcal glomerulonephritis (APSG), streptococcal toxic shock syndrome, and necrotizing fasciitis [1, 2]. In a study conducted in Turkey on the identification of GAS isolates at species level by 16S rRNA sequence analysis, S. pyogenes was found to be the most common when compared with gene bank data [3].

Many serious virulence factors play a role in the pathogenicity of S. pyogenes. The most notable factor is surface M protein encoded by the emm gene, showing its effect by facilitating adhesion, providing resistance to opsonophagocytosis and contributing to the overall burden of GAS infections [4, 5]. To date, more than 200 emm types and subtypes have been reported. The hyper-variable region of the emm gene forms the basis for emm typing in GAS. M protein is used as an epidemiological marker because of the different emm-type distribution in variable regions [6, 7]. emm gene sequence analysis is a useful tool to understand local transmission dynamics and geographical distribution of the emm types. Knowing the emm-type distribution of different patient populations in different regions of the same country can be used to understand local epidemiology and to determine local vaccine formulation. M protein, which is exhibited by all the strains of S. pyogenes, can be immunized in the host and is used in valuable vaccine development studies. Vaccines with 26-valves and 30-valves include streptococcal emm types that cause non-invasive (tonsillopharyngitis) and invasive infections [8, 9].

Superantigens (SAgs), containing mitogenic exotoxins, are produced by a small number of bacterial species and some viruses [10, 11]. GAS secretes several SAgs, e.g., streptococcal pyrogenic exotoxin (spe), streptococcal mitogenic exotoxin (smeZ), and streptococcal superantigen (ssa). These toxins are extracellular products and exhibit antigenic properties. SAgs, whose gene distribution and genomic heterogeneity have been determined, can be used as an additional epidemiological tool for further investigations of toxin-dependent diseases [12, 13].

The aim of the present study was to identify GAS isolates from 200 patients with tonsillopharyngitis between 2017 and 2018 in Canakkale and to detect emm types, exotoxin-gene profiles, and the coverage rate of the available vaccines.

Materials and Methods

Study Design and Sample Collection

All the experiments in this study were conducted in the Basic and Industrial Microbiology Laboratory of Biology Department at Canakkale Onsekiz Mart University. Each participant signed an informed consent form in accordance with the Declaration of Helsinki. Our study was approved by the Canakkale Onsekiz Mart University’s Clinical Research Ethics Committee (Project Number: 27/2017-E.33577).

200 patients with tonsillopharyngitis, who applied to Emergency, Family Medicine, Ear, Nose, Throat, and Child Health and Diseases Departments of Health Application and Research Hospital in Canakkale, from October 2017 to May 2018, were enrolled in this study. The throat swab samples were collected by the doctors. They were stored at 4 °C and transported to the microbiology laboratory within 2 h. Amies was used as a transport medium.

Identification and DNA Extraction of GAS

The throat swabs were streaked on 5% sheep blood agar plates (Bes-Lab, Turkey) and incubated under 5% CO2 at 37 °C overnight. β-hemolytic colonies were chosen and then subcultured. The isolates were identified using conventional methods (Gram staining, catalase, L-pyrrolidonyl β-NAPHTHYLAMİDE, bacitracin-SXT). All the isolates were stored at − 70 °C in Triptic Soy Broth (TSB; Merck, German) supplemented with 20% glycerol until further analysis. For DNA extraction from the GAS isolates, a commercial extraction and purification kit (Biospeedy, Turkey) was used according to the manufacturer's instructions.

Identification of GAS Species by 16S rRNA

In order to amplify a 1390-bp fragment of the target 16S rRNA gene, the primers listed in Table 1 were used [14]. PCR amplification was performed in a final volume of 25 µL containing 1× PCR buffer, 0.8 mM dNTP, 0.4 µM of each primer, 2 µL of template DNA, and 1 U/25 µL Taq polymerase. All the reagents were purchased from Geneon (German) and Biospeedy (Turkey). The amplification was performed on a thermocycler nexus gradient (Eppendorf) and the cycling program consisted of initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 55 °C for 45 s, annealing at 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR products were electrophoresed through 1.7% agarose gel.

Table 1 Primers, primers’ annealing temperatures (°C) and amplicon sizes (bp) used for amplification of 16S rRNA, emm, and SAg genes

emm Typing of S. pyogenes

The emm types of S. pyogenes isolates were determined by the Centers for Disease Control and Prevention (CDC, USA) website (https://www.cdc.gov/ncidod/biotech/strep/doc.htm) [15]. The set of primers, annealing temperature (Tm), and amplicon size (bp) are listed in Table 1. The products were run on 1.5% agarose gel.

Detection of Toxin Genes

Toxin genes of S. pyogenes, including speA, speC, speG, speM, speF, speB, speH, speI, speJ, speL, smeZ, and ssa, were detected by simplex and multiplex PCR with published primers as shown in Table 1 [16,17,18].

The amplification of all the SAg genes was performed using the following conditions: denaturing at 94 °C for 7 min, 30 cycles at 94 °C for 45 s, annealing at the appropriate temperature for 45 s standardized in the laboratory for each gene (Table 1), and 80 s of extension at 72 °C with a final elongation step at 72 °C for 7 min. PCR products were separated by electrophoresis on 2% agarose gel.

A 100-bp DNA ladder was used as a size marker (Biospeedy, Turkey). The PCR products were purified using PCR cleaning kit (Geneon, German) and then sequence analyses were conducted. The 16S rRNA, emm, and SAg gene sequences were searched for ≥ 99 homology by BLAST program, National Center for Biotechnology (https://www.ncbi.nlm.nih.gov/BLAST/).

Statistical Analysis

SPSS 22 version was used for statistical analysis. Gender, age, emm types of GAS, and superantigen genes were computed as data and analyzed by χ2 test. P value of < 0.05 was considered statistically significant.

Results

The DNA sequences of the 16 S rRNA gene region of the 15 GAS isolates were compared with the gene bank data and all were identified as S. pyogenes.

emm Types of GAS

According to the blast analysis results, six emm types, emm89 (5 isolates, 33.3%), emm44 (3 isolates, 20%), emm6 (2 isolates, 13.3%), emm84 (1 isolate, 6.7%), emm1 (1 isolate, 6.7%) and emm18.1 (1 isolate, 6.7%), were identified from 13 S. pyogenes isolates. emm typing could not be performed in two isolates.

Determination of Exotoxin-Gene Profiles by PCR

In this study, speG, speH, and smeZ were not detected among the 15 S. pyogenes isolates. speB (13 isolates, 86.7%) was the most prevalent, followed by speC (9 isolates, 60%), speF (5 isolates, 33.3%), ssa (4 isolates, 26.7%), speA (3 isolates, 20%), speM (3 isolates, 20%), speJ (2 isolates, 13.3%), speL (1 isolate, 6.7%), and speI (1 isolate, 6.7%). The exotoxin-gene sequences, which were found in the S. pyogenes isolates, were aligned with the multiple sequence aligner program at https://www.ebi.ac.uk/Tools/msa/Clustal Omega. The phylogenetic tree was drawn with NCBI clustal omega program and shown in Fig. 1.

Fig. 1
figure 1

Comparative phylogenetic tree of exotoxin gene species belonging to the 15 S. pyogenes isolates (A1–A15)

There was no statistically significant relationship between S. pyogenes emm types, exotoxin genes, and the genders and ages (P > 0.05) of the patients with tonsillopharyngitis (Tables 2 and 3). emm types and virulence gene distributions of the S. pyogenes isolates are presented in Tables 3 and 4.

Table 2 The distribution of emm types according to gender and age groups (< 15, 15 and > 15)
Table 3 The distribution of virulence genes according to gender and age
Table 4 Distribution of virulence genes in emm types

Discussion

S. pyogenes infection is observed in approximately 2–4% of 100,000 populations in developed countries and 12–83% in developing countries and in domestic populations of developed countries such as the USA and Australia [19]. 20.05% S. pyogenes were reported in Turkey, 29.5% in Iran, 9.2% in Nepal, and 7.5% in this study [20,21,22].

Molecular epidemiology studies report that there are significant differences in emm-type distribution at global level, especially between high-income countries and resource-poor countries and tropical regions. Although a small number of emm types are observed in developed countries, a wide variety of emm types are more common in disease-related strains in low-income countries. Previous studies have reported that socioeconomic factors have a considerable influence on the diversity of emm types and circulation of S. pyogenes [23].

In order to prevent the spread of GAS infections and post-streptococcal diseases, especially in individuals not receiving antibiotic treatment, two types of anti-GAS vaccines have been developed. These vaccines consist of peptides in the aminothermal region of the M protein. Based on the serotypes of GAS infections and the current epidemiological data in North America and Europe, the 26-valent and 30-valent recombinant multivalent vaccine, which contains 26 and 30 different emm types, has been designed. Epidemiological studies have shown that 26-valent vaccine is more effective in industrialized countries than in developing countries, accounting for an efficacy rate of 72% and 24%, respectively [9, 24].

In Taiwan, the most common emm type was emm12, followed by emm1 and emm4 [25]. The study reported that emm3 (80%), emm1 (16%), and emm75 (4%) were detected from throat cultures in Iran [26]. emm89 (16%), emm12 (10%), emm2 (9%), and emm1 (8%) types were common in Lebanon [27]. In Greece (2007–2013), emm genes were investigated in isolates collected from 1080 pharyngitis and 22 tonsillitis. In the pharyngitis emm12 (15.7%), emm1 (15.6%), emm4 (11.8%), emm77 (11.7%), emm28 (10.7%), emm3 (6.8%); and in the tonsillitis emm3 (18.2%), emm89 (13.6%), emm1, emm2, emm28, and emm4 types were detected. It has been reported that the recommended 30-valent GAS vaccine covers 97.2% of these emm types [28]. 7 of the 10 different emm types (emm1, emm5, emm14, emm18, emm19, emm29, and emm89) were detected in the 26-valent vaccine and the coverage rate was 50% [3]. In a study in our country, emm1 (30.9%), emm12 (14.6%), emm89 (8.1%), emm118 (7.3%), and emm4 (5.7%) were reported [20]. In the present study, 6 emm types were recovered from 13 S. pyogenes isolates. The most common emm type was emm89 (33.3%), followed by emm44 (20%), emm6 (13.3%), emm84, emm1, and emm18.1 (6.7%). As in the other studies, emm1 and emm89 types have also been detected in this study. emm1, emm6, and emm89 are available in 26-valent; and emm89, emm44, emm6, and emm1 types in the 30-valent vaccine. The isolates obtained from Canakkale were found to cover the 26-valent vaccine at a rate of 61.5% and the 30-valent vaccine at a rate of 84.6%.

Superantigens are toxins that can react in the host by activating T cells non-conventionally [29]. speA (17.2%), speB (72.4%), speC (13.8%), and speF (69.0%) exotoxin genes were detected in 29 GAS isolates in Taiwan [25]. In Lebanon, speB (87%), ssa (36%), and speG (30%) superantigens were commonly found [27]. In India, speB (100%), smeZ (100%), speC (28%), speH (28%), speI (28%), speL (22%), ssa (17%), speM (11%), and speJ (11%) were recovered from 18 GAS, isolated from throat cultures [30]. In Beijing, 13 SAg gene profiles were investigated in GAS, isolated from patients with pharyngitis. speB (99.2%), speC (99.2%), smeZ (99.2%), speF (98.8%), speG (98.5%), ssa (98.5%), speJ (49%), speA (48.6%), speI (46.3%), and speH (43.6%) were observed; speK, speL, and speM genes were not detected [31]. In a study in Turkey, speA (8.2%), speC (8.9%), and both genes (1.5%) were found in two isolates [20]. In the present study, smeZ, speG, and speH exotoxin genes were not found in any of the S. pyogenes isolates, differently from other studies. The most common virulence gene was found to be speB (86.7%), which was followed by speC (60%), speF (33.3%), ssa (26.7%), speA (20%), speM (20%), speJ (13.3%), speL (6.7%), and speI (6.7%). These rates were similar to other studies.

emm typing studies have been conducted on various populations to determine the epidemiology of S. pyogenes isolates and to provide information about the biology, pathogenesis, and genetic structure of bacteria. The absence of licensed vaccine and the increased resistance to antibiotics are a concern for public health. Several measures must be taken to prevent outbreaks caused by this pathogen [32].

The scope of studies should be expanded to include many regions in Turkey by increasing the number of samples and including invasive and non-invasive diseases caused by S. pyogenes.