Streptococcus pyogenes (group A streptococcus) is a Gram-positive bacterial pathogen. Extracellular surface molecules such as the M protein, hyaluronic acid capsule and fibronectin-binding proteins allow group A streptococcus (GAS) to adhere to, colonise, and invade human skin and mucus membranes under different environmental conditions [1]. GAS is the causative agent of acute bacterial pharyngitis, scarlet fever and impetigo [2] and is responsible for a wide variety of life threatening diseases in humans. The chronic infections lead to acute rheumatic fever (ARF) and rheumatic heart disease (RHD). In recent years, the GAS infections and associated complications are emerging in developing countries like India. More than 60 GAS virulence factors were identified, which were responsible for adherence to epithelial cells, internalization, invasion, systemic toxicity, immune modulation and evasion, cellular or tissue disruption and bacterial transmission [3]. The major virulence factor reported in GAS is the M protein encoded by emm gene. The M protein confers resistance to phagocytosis by polymorphonuclear leukocytes [4] and binds to complement control factors and thereby prevents the activation of alternate complement pathway [1]. The GAS produces serum opacity factor (SOF) both in extracellular and membrane-bound form [5], which is involved in GAS-host cell adherence process [6].

In 1935, group C streptococcus (GCS) and group G streptococcus (GGS) were first identified as human pathogens [7]. The infections with GCS and GGS can also cause acute pharyngitis, necrotizing fasciitis, sepsis, soft tissue infections and streptococcal toxic shock-like syndrome [8]. S. dysgalactiae subsp. equisimilis and S. anginosus are the two important species of GCS/GGS groups. Streptococcus dysgalactiae subsp. equisimilis can cause the whole spectrum of infections caused by S. pyogenes[9, 10]. A high rate of pharyngeal carriage of GCS and GGS in acute rheumatic fever (ARF) patients has also been reported [11].

GAS, GCS and GGS possess common virulence mechanisms including the expression of streptokinase, hyaluronidase, C5a peptidase and M protein [12]. Recently, the presence of GAS virulence genes encoding streptolysin S and glyceraldehyde-3-phosphate dehydrogenase has been reported in bovine S. dysgalactiae subsp. dysgalactiae (GCS) and human S. dysgalactiae subsp. equisimilis (GCS/GGS) [13]. Several reports indicated that GCS and GGS also possess emm or emmL genes [12, 14]. The existence of a considerable level of emm gene sequence homology between GAS and GCS/GGS groups has been reported [15]. The emmL gene polymorphisms have also been identified in human GGS isolates [6]. Several emm and emmL types of streptococci have been reported in India [16].

GAS produces many pyrogenic exotoxins, called streptococcal pyrogenic exotoxins (SPEs). These toxins act as superantigens (SAgs), which are potent T cell mitogens. Superantigens simultaneously bind to MHC class II antigens and T cell receptor (TCR) molecules and stimulate T cells to increase the levels of the cytokines TNF-α and IL-1β and T cell mediators like IL-2 and IFN-γ [17]. These cytokines in high concentration cause fever and shock [18].

The present study describes the prevalence of emm, emmL and SAg genes in the pharyngeal GAS, GCS and GGS strains isolated from a hospital in Tamil Nadu State, India. An attempt was made to determine the relationship between the superantigen profiles and emm types of pharyngeal streptococci. The strains that possess emm or emmL genes are referred to as typeable strains and the strains lacking emm or emmL genes are referred to as nontypeable strains. The presence of superantigen genes has been previously reported in typeable GAS, GCS and GGS strains. The presence of two GAS superantigens, streptococcal mitogenic exotoxin Z (smeZ) and streptococcal superantigen (ssa) has been reported in GGS emm types, st245 and stG480[19]. The presence of GAS superantigen, streptococcal pyrogenic exotoxin M (speM), is detected in GGS emm type, stG10 and GCS emm type, stC1400[19–21]. However, the presence of superantigen genes in nontypeable strains has not been previously reported. Here, we report the occurrence of different superantigen genes in typeable as well as nontypeable strains of pathogenic streptococci.

In the present study, a total of 124 strains were isolated from acute pharyngitis patients. Interestingly, 71.8% of them were emm and emmL nontypeable. The emm12 was the predominant emm type (28.6%) among the typeable GAS strains. The prevalence of emm12 was previously reported among the pharyngeal isolates of GAS from Chennai, South India [29]. The presence of emm1, emm3, emm28, emm12, emm4 and emm11 has been reported among the North Indian GAS isolates [30].

In this study, stG643.0 was the predominant emm type (28.6%) among GGS strains. In contrast, two other GGS emm types, stG485 and stGLP1, were previously reported to be prevalent among 49 GGS isolates from Chennai, India [31]. Other emm types previously reported from India [8] which also represented in this study were emm3 (2.9%) and stC46 (17.0%). In addition, the newly identified emm types include emm48.0 (5.7%), st3343.0 (2.9%), emm107.0 (2.9%), stS104.2 (2.9%), and emm30.11 (8.5%). According to CDC, these emm types have already been reported in United States, Mexico, Malaysia and New Zealand, but not from India. Therefore, further studies on the epidemiology of these emm types in India are needed.

The emergence of drug resistance among streptococci to macrolides (erythromycin and clarithromycin) and tetracycline are widely reported in recent years [32–34]. However, in the present study, all the streptococcal strains were susceptible to all the tested antibiotics including β-lactams, macrolides and tetracycline. Therefore, drug resistance is not a major threat among the streptococci isolated in this study population. However, untreated chronic infections may lead to severe complications such as acute rheumatic fever (ARF) and rheumatic heart disease (RHD).

SOF acts as an adhesin involved in the adherence of GAS to host cells [35]. It has also been reported that the interaction between SOF and the host extracellular matrix protein, fibulin-1, may be involved in the adhesion of GAS to extracellular matrices of the host [36]. Courtney et al. [37] reported that the SOF was expressed at a frequency of 50% among the clinical isolates of GAS. In contrast, only 14.3% of typeable GAS isolates were positive for SOF and all GCS and GGS strains were SOF-negative. Similarly, McDonald et al. [15] reported that most of the GCS and GGS strains of S. dysgalactiae subsp. equisimilis were SOF-negative. Therefore, SOF may not be the only factor responsible for the adhesion of GAS to the host and some other mechanisms might exist [37]. Some of the GAS adhesins reported other than SOF include lipoteichoic acid, hyaluronic acid capsule, vitronectin-binding protein and collagen-binding protein [2].

We have observed significant differences in the distribution of superantigen genes between typeable and nontypeable strains. The co-occurance of number of superantigen genes was relatively more in typeable than in nontypeable strains. Among 11 superantigens, a maximum of 6 superantigens (speC, speG, speA, speH, speI and smeZ) was detected in 17.1% of typeable strains, whereas this combination was detected in only 1.1% nontypeable strains. Totally, 26 superantigen profiles have been identified among the typeable and nontypeable strains. Of these, 23 profiles were detected in nontypeable strains and 9 profiles in typeable strains. Only six superantigen profiles were identified in both typeable and nontypeable strains. Other 20 profiles were confined to either typeable or nontypeable strains. As reported previously [38], the most prevalent chromosomally encoded superantingens, speG and smeZ, were detected in 97.1% and 77.1% of the typeable strains, respectively. In addition, these two genes were detected in 84.3% and 83.1% of nontypeable isolates, respectively. Similarly, Proft et al. [39] have reported the presence of these two superantigens in all the strains of GAS.

A high occurrence of GCS (59.7%) and GGS (25.0%) among pharyngeal streptococcal isolates was observed, which is in agreement with previous reports from Spain and Australia [10, 15]. It is known that more virulent forms of GCS and GGS could have emerged through the acquisition of superantigen genes from GAS [19]. In this study, many superantigen profiles have been identified among GCS and GGS strains and these strains could be considered as emerging pathogens.

Overall, 67 S. anginosus (52 GCS and 15 GGS) strains and 22 S. dysgalactiae subsp. equisimilis (16 GCS and 6 GGS) strains were nontypeable. Although the presence of superantigens has been reported in typeable GCS and GGS isolates, their presence in nontypeable strains has not been reported so far. However, in our study, compared to the strains of typeable S. pyogenes (GAS) and typeable as well as nontypeable S. dysgalactiae subsp. equisimilis, more numbers of superantigen profiles have been observed in nontypeable strains of S. anginosus (GCS/GGS). In a very recent study [13], the presence of GAS superantigens (speL, speC, speK and speM) has been reported in typeable S. dysgalactiae subsp. dysgalactiae (GCS) strains associated with bovine mastitis, but not in the human isolates of S. dysgalactiae subsp. equisimilis (GCS/GGS). We have identified the presence of superantigens in nontypeable strains of S. dysgalactiae subsp. equisimilis (GCS/GGS) associated with acute pharyngitis in humans. Therefore, our findings suggest that, like S. pyogenes, S. dysgalactiae subsp. equisimilis and S. anginosus may emerge as potential human pathogens. Some other factors might be involved in the pathogenesis of nontypeable strains in the absence of M protein. The presence of superantigens in nontypeable strains has not been reported so far. The role of superantigens in the pathogenesis of typeable GCS and GGS strains is well documented [2, 19, 20]. However, the role of superantigens in the pathogenic mechanisms of nontypeable strains is yet to be studied.

A high occurrence of GCS and GGS strains of S. anginosus and S. dysgalactiae subsp. equisimilis was observed among the acute pharyngitis patients and most of them were nontypeable. In addition, the presence of superantigens in nontypeable GCS and GGS strains was observed for the first time. Therefore, these strains may emerge as potential human pathogens. Though antibiotic resistance was not observed among these strains, earlier diagnosis and proper antibiotic treatment are required to cure acute pharyngitis and prevent from subsequent complications such as acute rheumatic fever (ARF) and rheumatic heart disease (RHD).