Background
Streptococcus pyogenes or Lancefield group A
Streptococcus (GAS) is responsible for a diverse range of clinical manifestations. Acute infection causes pharyngitis, impetigo, scarlet fever, cellulitis, acute bacterial endocarditis and necrotizing fasciitis. Ineffective or delayed treatment, and infection with drug-resistant bacteria eventuates in chronic immune-mediated disorders such as acute rheumatic fever (ARF) and acute glomerulonephritis (AGN) with attended morbidity and mortality worldwide [
1‐
6].
The fast emergence of antimicrobial resistance amongst
S. pyogenes therefore needs molecular characterization of the newly variant strains. The conventional M serotyping is cumbersome, less effective and demand preparatory requirements [
7,
8]. A large number of
S. pyogenes isolates remains M untypable by the available present range of high titre antisera [
9‐
11]. Alternative techniques used for
emm and
vir typing are favored.
emm typing is based on sequence analysis of
emm gene [
8], that encodes for the major virulence factor M and M-like proteins while
vir typing is based on amplification of virulence regulon-specific polymerase chain reaction (PCR) amplicon followed by restriction fragment length polymorphism (RFLP) [
12].
Different reports have shown a fair prevalence of GAS infections among children [
6,
13,
14] and strain variations among the GAS isolates [
15,
16]. A sudden world-wide increase in antimicrobial resistance in
S. pyogenes has been observed during the last few years [
17,
18]. Macrolide resistance among GAS has been widespread [
19,
20] possibly linking to the spread of a particular clone by selection pressure due to random usage of macrolides [
21]. Recent reports on tetracycline resistance identify the ineffectiveness of this drug against infections with GAS [
22‐
26]. Streptococcal pyrogenic exotoxins act as super antigens and stimulate lymphocyte activation to induce fever and extensive proliferation with the consequent massive release of cytokines [
27]. Fibronectin binding proteins are considered as major Streptococcal adhesins and amongst them fbp54 is expressed in human host environment [
28].
Reports on Streptococcal pyrogenic exotoxin genes and fibronectin binding protein gene expression in noninvasive infections are relatively few from India. The present study tries to establish the correlation of the GAS genotypic variation with the changes in molecular patterns of virulence regulon, antibiotic resistance and expression of pyrogenic exotoxins and fibronectin binding protein genes.
The isolates have been characterized with
HaeIII,
HinfI and
XhoI restriction fragment length polymorphism to evaluate their applicability in
vir typing analysis following methodology by Gardiner et al. [
9,
12].
Discussion
In the present study, the
emm and
vir type diversity among GAS isolates obtained from different patients was analyzed. Reports by earlier workers documented 57.5, 19.71 and 40% heterogeneity with
emm49,
emm77 and
emm74 genotypes to be the most prevalent types respectively [
15,
34,
35]. In another study, 32.35% heterogeneity was reported [
16]. A Norweigian study revealed 44% variation with
emm1 to be the most prevalent genotype [
36]. In a recent outbreak of GAS infections, the most uncommonly reported
emm type 58 was shown to be most frequent isolate [
37].
In present study, emm typing resulted in 14.28% heterogeneity in the isolated GAS strains which is indicative of lower strain variation in this context. The most detectable type found was emm49 (17.85%). Thus different values of heterogeneity in the isolates were observed by the present and earlier investigators, might be due to variation of prevalent strains in different geographical regions having different climatic conditions. It could possibly be predicted that the genetic heterogeneity could be temperature and humidity dependent, however this fact requires a complete different investigation. The results of this study demonstrated some emm types and their subtypes, like emm81/emm81.1, emm25/emm25.1 and emm1/emm1.1, emerged due to point mutation, reflecting in the origin of N-terminal emm sequence variability and implying the importance of emm strain typing (sequencing data not shown).
As children are more susceptible to GAS infections [
38‐
40], and as associated
emm-types and clinical presentations are influenced by population immunity and strain tropism [
41], the
emm distribution patterns in relation to patient’s age, the children in particular, are significant. In this study occurrence of
S. pyogenes infection was found average. As children are more susceptible to GAS infections with attendant life-threatening remote sequelas (RHD and AGN) as well, it is believed that the dominance of
emm49 as well as other frequently occurring
emm types in infant and children needs an independent and systematic study on association of immunological parameters with
emm types.
Vir typing measures the changes in the region of the genome that determines major virulence factors that are involved in anti-phagocytic activity, attachment, colonization and in avoiding recognition by the immune systems [
12]. The
HaeIII vir typing patterns in the present study were found dissimilar to other reported typing patterns. So we assigned our own VTs to analyze the typing patterns of the isolates from eastern region of India. We screened 13
vir types from 140 isolates by
HaeIII digestion, which accounted 9.28% diversity in GAS isolates. Here we observed that
vir types were entirely different from the reported typing patterns of Australia, Romania and northern India.
HaeIII vir typing patterns of Australian GAS isolates produced 4 to 8 fragments ranging from 200 bp to 4 kb, where 1.4 kb, 500 bp, and 275 bp were found common in the isolates tested [
9].
vir typing patterns of Romania yielded 2 to 8 fragments ranging from 100 bp to 4.2 kb with a common fragment at 1.2 kb [
42]. Reports from north India showed 4 to 6 fragments varying from 300 bp to 5 kb where 300 bp, 550 bp and 1.2 kb were common for the majority of isolates [
15]. We found 3 to 7 fragments of 200 bp to 4 kb and a 1.2 kb fragment was found common to all isolates. The majority of isolates shared the fragments of around 200 bp (81.42%) and 550 bp (87.85%). On comparing our present findings with those previous observations, a 1.2 kb fragment was found common in most of the study regions (except Australian typing patterns). In India, in addition to 1.2 kb, a 550 bp was also found common to the majority of isolates. Thus, these two segments of the virulence regulon of
S. pyogenes remain conserved in Indian strain types.
The overlaps between
emm and
vir typing showed that one specific
emm type was always represented by one particular
vir type whereas one
vir type was shared by more than one
emm types. However, in two cases, different VTs were obtained for isolates of one
emm type and its subtype. This finding supports the concept that the heterogeneity demonstrated by
vir typing is primarily due to variations among
emm genes rather than diversity in the architecture of the
vir regulon [
9] and thus, an insertion or deletion of one or more nucleotide not only categorizes a specific
emm type into its subtypes but also changes the expression of its virulence regulon. Thus
vir typing analysis is found more informative than
emm typing, regarding architectural variation of virulence regulon conferring the virulence on
S. pyogenes.
Furthermore the use of the second restriction enzyme
HinfI appeared to be very useful in discriminating
HaeIII typed isolates, which supports the concept of Gardiner et al. [
9]. In the present study, though
HinfI was found proficient in distinguishing the isolates of the same
HaeIII vir types and also generate more fragments comparing
HaeIII but it did not generate any conserved fragments, unlike
HaeIII digestion. Therefore, successive use of these two enzymes can be a tool for
vir typing analysis.
XhoI digestion produced fewer fragments and was less informative.
In order to study the strain characteristics contributing to virulence patterns, the isolates were evaluated for the presence of different toxin and fibronectin binding protein genes. Extracellular proteins that reportedly play a role in the pathogenesis of GAS are represented by streptococcal exotoxins, a few of which have superantigenic properties that allow them to activate large subsets of T cells, with massive cytokine release as a consequence [
31]. Previous report from India showed lower incidence of
speA gene (5.8%) and higher incidence of
speB and speF gene (92.3, 86.5%) in noninvasive infection [
43]. In contrast, the current study showed
speA gene less contributing for GAS infections, whereas the observed predominant occurrence of
speC and
speF genes strongly suggest their association with incidence of infections. Association of
SpeB and
fbp-54 genes was noticed in less number of cases and probably partly contributes towards virulence. It is of considerable interest to study the correlation of these exotoxin gene expressions with cytokines activation in this region by further study.
Another important aspect of this study is GAS antibiotic sensitivity patterns and their correlation with
emm genotyping. Penicillin has been used routinely for treating GAS infections for over 50 years, and yet GAS has remained penicillin susceptible [
44,
45]. Since penicillin may be given at higher doses without ill effects, the observed intermediate sensitivity in our study may have less impact. A study from north India [
7] showed diminished susceptibility in 20.6% of strains which is in support of our observation but contrast to previous reports of 100% susceptibility [
46,
47]. Erythromycin resistance and therapeutic failure have been observed to be on the rise worldwide due to their increased medicament [
48‐
50]. In this study, sensitivity to erythromycin was found very high. The observed erythromycin resistant cases were though small in number, but alarming in this region as these were obtained mostly from children, and erythromycin is the 1
st alternative drug of choice for penicillin hypersensitive patients as well. Interestingly, all four erythromycin resistant strains were of genotype
emm49, suggesting that the genotype’s association with the emerging erythromycin-resistance in this region. Association of erythromycin resistance and
emm typing has been documented earlier.
emm12 from Korea and Japan [
51],
emm12 and
emm75 from Pittsburgh [
52],
emm90 from Hawaii [
53],
emm 4,
emm 28, and
emm 77 from Western Greece [
54] showed strong association with erythromycin resistance. In present study, association is different from earlier workers as
emm49 was constantly associated with the erythromycin resistance. In spite of clindamycin resistance report [
55] earlier, all isolates of present study were found sensitive to clindamycin and vancomycin. High tetracycline resistance among
S. pyogenes isolates have been reported from different parts of the World [
22,
44,
56,
57]. In the present study, high percentage of resistance was shown by tetracycline, which is in corroboration with the previous findings. 14 and 28 different tetracycline-resistant
emm types were identified in studies from Spain and Tunisia, respectively [
58,
59]. Present study identified 75 tetracycline-resistant isolates, belonging to 12 different
emm genotypes. These findings indicated that for study of different regions,
S. pyogenes strains are approaching tetracycline-resistance, attributable to modifications in
tet (M) gene commonly associated with the conjugative transposon Tn916 [
59].