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.