Background
Streptococcus pyogenes, or Group A Streptococcus (GAS), is an exclusively human pathogen responsible for a broad variety of clinical manifestations ranging from pharyngitis and impetigo to invasive diseases, such as necrotizing fasciitis and toxic shock syndrome. Some strains can also trigger autoimmune diseases, such as acute rheumatic fever, rheumatic heart disease and glomerulonephritis [
1]. GAS infections are the major cause of morbidity and mortality worldwide. The prevalence of severe GAS diseases is at least 18.1 million cases, which cause approximately 517,000 deaths per year [
2].
M protein is a surface component of GAS and one of the main virulence factors due to its anti-phagocytic properties [
3]. This protein contains a hyper variable amino terminal end that serves as substrate for gold standard
emm-typing for strain identification. More than 220 different
emm-types have been described [
4]. Systematic epidemiological reviews clearly highlight significant differences in
emm-type distribution across different regions of the world. Relatively limited numbers of
emm-type are recovered from high-income settings, while a much higher diversity of strains circulates in low-income settings [
5,
6]. A complementary typing system,
emm-pattern typing, is based on the presence and arrangement of
emm and
emm-like genes located in the
mga locus within the
S.pyogenes genome. This classification is correlated with tissue tropism as follows: A-C
emm-pattern isolates are usually recovered from the throat infections, D
emm-pattern strains are usually isolated from the skin (impetigo), and E
emm-patterns are recovered from both biological sites [
7,
8].
Sanderson-Smith et al. recently proposed a functional classification of the
emm-types in clusters according to the phylogenetic origin and microbiological characteristics of the strain. The cluster classification enabled comparison between strains and serves as a tool for vaccine development [
9].
GAS contains numerous genes encoding virulence factors, such as streptococcal pyrogenic exotoxins (Spe proteins). These proteins constitute a family of bacterial toxins with powerful mitogenic effects on T cells expressing a particular Vβ domain of the T cell receptor molecule, inducing non-specific polyclonal activation of the immune system by binding directly to class II MHC molecules [
10]. Several studies have reported that Spe exotoxin content is correlated with
emm-types and associated with clinical manifestations [
11‐
13]. Spe exotoxins most likely contribute to the severity of GAS infections. However, the exact molecular mechanism involved in specific pathologies is still not understood [
14].
To date, no anti-streptococcal A vaccine is available; however, several candidates based on both N- and C- terminal portions of the M protein are in different stages of development [
15]. Briefly, the 30-valent is based on the highly variable amino-terminal region of the M protein [
16], and the J8 candidate vaccine a construction of minimal B-cell epitope from the C-repeat region [
17].
StreptInCor candidate vaccine is based on amino acid sequences of the conserved region of the M5 protein. This candidate vaccine, in contrast to the others, contains both B and T cell epitopes to provide a strong protective immune response [
18].
Although GAS infections are common in several regions of Brazil, only a few studies on the prevalence,
emm-type profiles and virulence factors of the strains are available [
19‐
21]. Here, we described the
emm-type and superantigen profile of the most prevalent strains in Sao Paulo and assessed the theoretical coverage vaccine.
Discussion
Streptococcus pyogenes is an important human pathogen responsible for several invasive and non-invasive diseases in Brazil and worldwide. In this study, we characterized 229 invasive and non-invasive
Streptococcus pyogenes samples from patients treated at the Clinical Hospital in Sao Paulo, Brazil. Great diversity of
emm-types was observed. Forty-eight
emm-types were observed in the 229 samples, with the 10 most frequent
emm-types accounting for 69 % of all isolates. In terms of GAS strain diversity, a Simpson Reciprocal Index of 1 corresponding to a theoretical situation where only one
emm-type/cluster has been recovered, representing the lowest diversity possible. The maximum value of the Simpson Reciprocal Index corresponds to the total number of
emm-type/cluster recovered in one area. The higher the value is, the greater the diversity. The reciprocal Simpson index of diversity found in this study was relatively low (12.7) when compared to the index of 26.72 for Brasilia (in the central region of Brazil) [
19]. On the other hand, our results were similar to those reported for high incomes suburbs from Salvador, in northeastern Brazil [
20].
The distribution of the strains identified in this study is comparable to those found in other countries, particularly in high-income countries in Asia, the Middle East and Latin America, in which
emm1 and
emm12 were the most common types, as reviewed by Steer [
6]. Interestingly,
emm1,
emm12 and
emm89 have also been found in various studies conducted recently in several countries in Europe and China; these types were frequently correlated with invasive and/or noninvasive isolates [
25].
emm77 had a high frequency in the invasive isolates found here. In addition, this strain has been associated with non-invasive diseases in Germany [
26] and was found in both invasive and non-invasive isolates in Spain [
12]. Among the 229 isolates, E and A-C
emm-patterns were found in similar proportions, whereas pattern D was less frequent. Interestingly, studies from Brasilia, in the Central region of Brazil [
19], revealed a higher proportion of E and D patterns (51 % and 36 %, respectively), whereas A-C patterns was rarely observed (9.5 %). The data demonstrate the variability of streptococcal strains in Brazil, which may be related to socio-economic differences and can be extended to other countries in which there are also social disparities.
Other factors that play a role in the clinical manifestation of S. pyogenes infection may be due to the associations between emm-types and superantigens.
In this study, the chromosomally encoded genes
smeZ and
speG occurred at high frequency in nearly all isolates (95.6 and 88 %, respectively); both were present in all
emm-types at high frequencies (<70 %), except
speG in
emm77(43 %), in according with a variety of others studies [
12,
27‐
29].
The other chromosomal gene,
speJ, was present in only 35 % of isolates and was absent in diverse
emm-types, similar to others studies [
12,
29,
30].
Among the phage-encoded genes,
speC was the most prevalent, detected in 48 % of the isolates, followed by
ssa (27 %),
speA (19 %),
speH (16 %), and
speI (14 %). The
speC,
ssa,
speH and
speI genes presented similar frequencies to those found in others studies, whereas the
speA gene generally had a lower frequency in our samples [
25,
30,
31].
speA was present in
emm3 (100 %),
emm1 (62 %) and only one sample of
emm6. The
speA genes has been commonly detected among 1 isolate in several studies [
32].
Currently, no anti-streptococcal vaccine is available in animal models of streptococcal disease, despite extensive efforts. Some models of anti-streptococcal vaccines are in different stages of development. Among them, the 30-valent contains short peptides from the highly variable amino-terminal region of the M protein [
16], and the J8 vaccine candidate comprises a 12 amino acid minimal B-cell epitope from the C-repeat region flanked by 16 amino acids of a yeast DNA-binding protein conjugated to the diphtheria toxoid [
17].
The vaccine candidate developed by our group, called StreptInCor, is based on the M5 protein C-terminal region [
18], specifically the C2 and C3 region that is conserved among serotypes. Through
in silico analysis with predicted amino acid sequence alignment, StreptInCor candidate vaccine had high sequence identity with 46 of the 48
emm-types described here (identity ranged from 94.5 % to 59.7 %, mean of 71 %), which is an important property for the probability of protection. In previous data, we described the structural, chemical, and biological properties of the StreptInCor peptide and demonstrated that the molecule is stable, which is an important property for a vaccine candidate. The possibility of the StrepInCor vaccine candidate epitope being processed by antigen-presenting cells (APCs) generating diverse peptides has also been previously demonstrated. The approach resulted in the observation that the vaccine epitope could be recognized by any individual, thus enabling a broad coverage capacity to trigger specific immunity [
33].
The efficacy of this vaccine in animal models was evaluated in inbred and outbred mice, and a strong humoral response with high IgG production was observed [
18]. Immunized Swiss mice challenged with the
emm1 strain had a survival rate of 87 % at 21 days compared with lower survival in controls (53 %) [
34].
Similar results have been observed in HLA class II transgenic mice, which also presented a specific and long-lasting immune response without developing deleterious reactions after one year. These results indicated that StreptInCor is a safe candidate vaccine [
35].
In addition, the four most common
emm-types included here (
emm1,
emm12,
emm22 and
emm87) were opsonized by StreptInCor-induced antibodies [
36]. The strains identified here were fit into 12 of the 19 different
emm-clusters and exhibited diverse phylogenetic origin and consequently different mechanisms of infection and resistance to escape the host immune system, supporting the hypothesis that StreptInCor vaccination would likely protect against infection caused by strains from different
emm-clusters.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SF and KMA contributed equally to the study design, coordination, analysis and interpretation of data, and drafting of the manuscript; SF, KMA, RA, and AT carried out the lab work for strain characterization and maintenance; RA and EP contributed to analysis and interpretation of data and drafting of the manuscript; PRS carried out the sequence alignments and the theoretical vaccine coverage capacity statistical analysis; FR and JAJ carried out the sample collection and microbiological assays for S. pyogenes diagnostics; LG contributed to study design, data analysis and interpretation, and drafting and revising the manuscript; JK contributed to study design and drafting and revising the manuscript. All authors have read and approved the final manuscript.