Background
Streptococcus pyogenes (Lancefield group A streptococcus; GAS) is a major pathogen causing infectious diseases in children. It causes suppurative and non-suppurative diseases, such as erysipelas, suppurative tonsillitis, scarlet fever, rheumatic fever, and glomerulonephritis [
1]. Globally, there are about 616 million cases of GAS pharyngitis every year, among which, 17,800 cases are new infections, and about 517,000 patients with severe GAS are deceased every year [
2]. Recently, the positive rate of GAS was estimated at 21.2% in the pharyngeal culture of patients diagnosed as “streptococcal infection/tonsillitis/angina” [
3]. Moreover, the incidence of streptococcal pharyngitis is common in children aged 0–14 years. From 2012 to 2014, the average number of positive cases of streptococcal culture was 2685.1/100,000 children in Beijing, including 1652.7 outpatient visits [
4]. In 2011, scarlet fever broke out in mainland China and Hong Kong, with a sharp increase in incidence [
5,
6]. Penicillin is the preferred clinical treatment of GAS infection, while erythromycin is the first alternative antibiotic for patients allergic to penicillin, followed by clindamycin. The resistance rate to macrolidesis gradually increasing, which might be related to the overuse of such antibiotics [
7,
8]. In our previous study, 95 isolates were recovered from suppurative tonsillitis, acute glomerulonephritis, scarlet fever and streptococcal dermatitis. The resistance rates of the isolates to erythromycin, clindamycin and tetracycline were 98.9, 100, 94.7% respectively [
9].
The M protein encoded by the
emm gene is the main pathogenic factor of GAS, and different types vary in pathogenicity. Therefore,
emm typing is employed to track outbreak and routinely monitor GAS diseases. From 2011 to 2013, the proportion of
emm12.0 in children with GAS infection in Xicheng District of Beijing decreased gradually, and
emm1.0 increased every year [
10]. The interplay among GAS diseases,
emm types, superantigen gene, and antimicrobial resistance needs further investigation [
11,
12]. The resistance of GAS to macrolides is related to the mechanism underlying target modification mediated by
ermA and
ermB and the pumping mechanism mediated by
mefA. Intriguingly, different primary mechanisms of resistance regulate various epidemic strains [
13]. The superantigen genes are the main virulence factors and closely related to the pathogenicity of GAS. Hitherto, 11 superantigen genes, including
speA,
speC,
speG,
speH,
speI,
speJ,
speK,
speL,
speM,
smeZ, and
ssa, were found to be distributed among various strains [
14,
15]. Previously, the
emm typing and superantigen distribution of GAS strains from different regions of mainland China were investigated, which indicated that the typing and antimicrobial resistance were slightly different [
16,
17]. In this study, we recovered GAS isolates from patients with scarlet fever in Children’s Hospital, Capital Institute of Pediatrics from 2016 to 2017, and conducted antimicrobial susceptibility test,
emm genotype analysis, and combined analysis of superantigen to assess the molecular epidemiological characteristics and antimicrobial resistance mechanisms of GAS strains.
Discussion
S. pyogenes or GAS is a leading pathogen causing infectious diseases in children. The GAS infection manifests as mild non-invasive diseases, such as acute pharyngitis or life-threatening invasive diseases, such as sepsis and toxic shock syndrome [
15]. Scarlet fever is a acute infectious disease caused by GAS, that can affect people of all ages, but it is most often seen in children. Before the advent of antibiotics, scarlet fever was extremely serious, often causing long periods of illness, many dangerous complications, and even death. A re-emergence of scarlet fever has been noted in Hong Kong, mainland China, South Korea, and England, UK, and other countries around the world since 2008 to 2014 [
21‐
23]. Penicillin has always been the preferred treatment for the GAS infection. In penicillin allergic patients, macrolides are the most commonly used antibiotics for treating streptococcal infections. However, the resistance rate of macrolides has also been increasing gradually [
7]. Of the GAS isolates recovered from the throat swabs of children with pharyngitis in Madison, Wisconsin, 15% demonstrated nonsusceptibility for clindamycin and erythromycin, and inducible resistance (positive D-test) was detected in 12% isolates [
24].
S. pyogenes isolates collected from infected patients from 7 cities/provinces in China during the years 2009–2016, were phenotypically susceptible to penicillin, ampicillin, cefotaxime, and vancomycin, whereas 93.5, 94.2, and 86.4% were resistant to erythromycin, clindamycin, and tetracycline, respectively [
25]. In this study, the GAS isolates recovered from children with scarlet fever were highly sensitive to penicillin, cephalosporin, levofloxacin, and vancomycin while the resistance rates to erythromycin, clindamycin and tetracycline are 98.3, 96.6 and 90.23%, respectively (see Table
3). No significant shift was detected in the resistance rate of GAS isolates to antibiotics between 2016 and 2017. These findings were consistent with those from previous study in 2013 that the resistance rates of isolates obtained from scarlet fever in Beijing to erythromycin, clindamycin and tetracycline were 99.3, 99.3 and 88.2% respectively [
26]. However, Erythromycin resistance was found in 51.4% of isolates in India [
27]. In Brazil, resistance to erythromycin and clindamycin was 15.4% [
28]. Thus, antimicrobial susceptibility test is suggested before choosing erythromycin as an alternative treatment for penicillin-allergic patients.
Traditionally, GAS infection patients with penicillin allergy are commonly treated with macrolide antibiotics. In the late 1990s, the resistance rate of GAS isolates to erythromycin in most regions of China was less than 50%. Around 2008, the resistance rate of GAS to erythromycin was 95–100%, while that for the isolates in Taiwan decreased from 53.1% in 2000 to 0% in 2010, but rapidly increased to 65% in 2011. The genes involved in erythromycin resistance were
mefA (53.1%),
ermB (35.9%), and
ermTR (10.9%) [
29]. In this study, the resistance rate to erythromycin was 98.3%, that was much higher than that detected in North America and some European countries (9.6–35.8%). Of the total 297 isolates, 290 (97.64%) harbored the
ermB gene, 5 (1.68%)harbored
mefA, none harbored
ermA. This phenomenon differed from that in the USA, Italy, Chile, and Canada where erythromycin-resistant strains of GAS are mainly M-resistant phenotypes mediated by
mefA. The target modification mechanism mediated by
ermB is the main resistance mechanism of GAS in China. The pattern of antibiotic resistance fluctuates worldwide. In a study in India, 51.4% of the GAS isolates were resistant to erythromycin, of which, 65.1% harbored
ermB and 32.5% harbored
mefA as the only genes resistant to macrolides, while 2.2% harbored both
ermB and
mefA [
8]. The resistance rate of erythromycin and clindamycin in Korea decreased from 51.0 and 33.7% in 2002 to 9.8 and 8.8% in 2004, respectively. The sharp decline in erythromycin resistance in a short period may be related to the change in
emm type distribution in the community [
30]. In Portugal, the resistance rates of erythromycin and clindamycin were 14% (carrying the
ermB gene) and 9% (harboring the
ermTR gene) in 2010–2015, respectively [
31]. Thus, it could be deduced that the high resistance rate of macrolides in China was related to the distribution of
emm types.
The distribution of
emm genotypes of GAS varied according to the countries, regions, and periods.
emm1 is the most popular type in Germany, consistent with that in the USA, Australia, and Japan; the prevalent types were
emm1 (31.8%),
emm28 (15.4%), and
emm 89 (14.5%) [
14,
32]. Presently, the most popular
emm types in China are
emm12 and
emm1. In 2011, two patients with scarlet fever died in Hong Kong; the GAS pathogens were
emm1 and
emm12 [
33]. In Chaoyang district, Beijing, in 2011, the main GAS epidemic strain of scarlet fever in children was
emm12.0 [
34]. In our study, 297 GAS isolates were recovered from patients with scarlet fever at the Children’s Hospital from 2016 to 2017. Nine
emm types, including 28 subtypes, were identified, of which,
emm12 (65.32%, 194/297) and
emm1 (27.61%, 82/297) were the most prevalent
emm types (Table
4). In a previous study, eight
emm types were identified in 155 isolates of GAS recovered from the pharynx of children with scarlet fever, pharyngeal tonsillitis, as well as healthy carrier in Beijing.
emm1.0 and
emm12.0 were the main types of scarlet fever and pharyngeal tonsillitis.
stg485,
emm18.0,
emm1.0, and
emm12.0 were the main types of healthy carrier [
35]. From 2009 to 2016, the main
emm types of GAS strains were
emm12 (42.9–62.2%) and
emm1 (30.7–35.0%) [
25,
36]. Interestingly, the proportion of
emm12 and
emm1 in this study was similar to that reported previously. These results showed that the
emm genotypes of GAS isolates changed significantly in recent years as compared to those identified in the 1990s. The most common
emm genotypes in 1993–1994 were
emm3.1,
emm1.0,
emm4.0, emm12.0,
st1815.0,
emm6.0, and
emm18.0 [
37]. You Y collected 2484 strains of GAS during 2011–2018 and found that the prevalent
emm types of GAS causing scarlet fever shifted for 8 years in Beijing since 2012, the frequency of
emm12 S pyogenes started to decline from 2011, whereas
emm1 started to increase and then exceeded
emm12 in 2013 and 2014. Since 2015,
emm12 exceeded
emm1 and became the main type again. Notably, numbers of nonpredominant types
emm128 increased substantially in 2017 and of
emm3 in 2018 [
38].
The main types of GAS in China are different from those in other countries around the world. A total of 35
emm types in 1282 isolates from GAS infection in children in Greece from 2007 to 2013, included
emm1 (16.7%),
emm12 (13.6%),
emm77 (10.9%),
emm6 (6.8%), and
emm89 (6.6%) [
1]. Among 1122 invasive isolates from Finland during 2008–2013, 72
emm types were identified, of which
emm28 (26%),
emm89 (12%), and
emm1 (12%) were the most common types [
39]. The main
emm types of iGAS strains in Portugal from 2010 to 2015 were
emm1 (28%),
emm89 (11%),
emm3 (9%),
emm12 (8%), and
emm6 (7%) [
31]. Furthermore, the isolates of
emm60.1 and
emm63.0 genotypes were prevalent in the children from the villages of Guizhou Province in China, which led to the outbreak of acute glomerulonephritis in 2005 [
40]. In 2012, many people suddenly had a fever, sore throat and/or fatigue, headache, and other similar symptoms within 24 h in Beijing. The isolated GAS strain had the same genotype (
emm89), which was first discovered to cause tonsillar pharyngitis in Beijing, China [
41].
emm89was also identified in this study. Between January 2016 and May 2017, a rare outbreak of GAS, caused by
emm66.0, occurred in England and Welsh [
42]. The local outbreak of GAS infection is related to the shift of
emm types. Moreover, different
emm types carry different resistance genes, which leads to the difference of erythromycin resistance rate. In erythromycin-resistance isolates in Brazil the
ermB gene was predominant, followed by the
ermA gene. Thirty-two
emm types and subtypes were found, but five (
emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates [
28]. These results were different from that in China [
21]. Therefore, continuous monitoring of streptococcal infection is required.
GAS superantigens, except
speG,
speJ, and
smeZ encoded by chromosome,
speA,
speC,
speH,
speI,
speK,
speL,
speM, and
ssa are encoded by phage, which is the main driving force for pathogenic strains to obtain pathogenic factors through transfer. The transfer and mutation of genes can produce highly pathogenic GAS strains, which affect the epidemic situation of the GAS disease, resulting in different distributions of the
S. pyogenes superantigen gene spectrum in different periods and geographical areas. A study from Portugal showed that
smeZ (96.0%) and
speG (86.9%) were common in GAS, followed by
speC,
ssa,
speJ,
speA,
speK, and
speI [
43]. A multicenter study in China has proved that 31.1% of the GAS isolates harbored
speA, while 58.6% harbored
speC [
17].
emm1 and
emm12 were consistently the most prevalent types of GAS isolates from pediatric patients during 1993–1994 and 2005–2006. Isolates carrying six or more superantigen genes increased from 46.53% in 1993–1994 to 78.39% in 2005–2006. The level of
ssa,
speH, and
speJ genes increased, while that of
speA decreased. The gene profiles of superantigen were associated with the
emm type, but strains of the same
emm type occasionally carry different superantigen genes in the two periods. Intriguingly, no significant difference was detected in the distribution of
emm types and
SAg gene profile between isolates from different diseases [
37]. In this study, 11 superantigens were detected in GAS isolates, and
speC,
speG, and
smeZ were the most common superantigens.
emm1 harbored
speA,
speC,
speG,
speJ,
speM,
ssa, and
smeZ, but the content of
speI,
speK,
speL was less.
emm12 type tended to contain
speC,
speG,
speH,
speI,
speM,
ssa, and
smeZ, with little or no
speJ,
speK, and
speL. A study from Germany showed that the most common superantigen genes in GAS were
speG (92.1%),
speJ (50.9%), and
speC (42.0%). Simultaneously, it was observed that
emm types or superantigen genes had significant correlation with clinical complications [
14]. In an outbreak of GAS infection caused by a rare
emm58 type in a multiple trauma treatment center, it was found that the strain was macrolide and tetracycline resistant and produced the Streptococcal exotoxins
speB (a streptococcal cysteine protease)
, speC, speG, smeZ and speF (now considered to be a DNase) [
44]. From 2009 to 2016, all isolates from infected patients in 10 general tertiary hospitals in 7 provinces (cities) of China, whether invasive or no-invasive, harbored genes for the protease
speB and the pore-forming toxin
slo. The other virulence genes,
smeZ,
speF, and
speC accounted for 96.4, 91.4, and 87.1% of collected isolates, respectively. All strains were sensitive to penicillin, ampicillin, cefotaxime, and vancomycin, whereas the resistance rates to erythromycin, clindamycin, and tetracycline were 93.5, 94.2, and 86.4% respectively, which indicated high genotype diversity and high prevalence of macrolide resistance of
S. pyogenes in clinical isolates circulating in China [
25]. In the previous studies on children, 30.5 and 57.2% of GAS isolates harbored superantigen genes
speA and
speC, respectively. 88.8% of
emm1.0 genotype strains contained the
speA gene, while 69.6% of the
emm12.0 genotype strains contained the
speA gene [
17]. In Taiwan, isolates with
emm1.0,
emm4.0, and
emm12.0 genotype were the main causes of non-invasive diseases. A few isolates with
emm1.0 genotype harbored
speC and
speH genes, while a few isolates with
emm12.0 genotype harbored
speJ and
smeZ genes [
45]. In Spain, the isolates of
s.pyogenes with
emm1.0 genotype, associated with pharyngitis, carried
speA, speG and
speJ genes, but did not carry
speC, speH, speI or
ssa genes [
46]. All the above studies showed that the distribution of
emm genotypes and superantigen gene profiles were time and region dependent.