Molecular epidemiology and antimicrobial resistance of group A streptococcus recovered from patients in Children

Background: Group A streptococcus (GAS) is an important human pathogen responsible for a broad range of infections. Epidemiological surveillance has been crucial to detect changes in the geographical and temporal variation of the disease pattern. The objective of this study was to investigate the molecular epidemiological characteristics and antimicrobial resistance of GAS isolates from patients in Children’s Hospital in Beijing. Methods: From 2016 to 2017, pharyngeal swab samples were collected from the outpatients in Children’s Hospital, Capital Institute of Pediatrics, who were diagnosed as scarlet fever. Antimicrobial susceptibility test was performed according to the distribution of common antibiotics and Clinical and Laboratory Standards Institute (CLSI) recommendations. The distribution of the macrolide-resistance genes ( ermB, ermA, mefA ), emm (M protein-coding gene) typing, and superantigens (SAg) gene profiling were examined by polymerase chain reaction (PCR). Results: A total of 297 GAS strains were collected. The sensitivity of the strains to penicillin, ceftriaxone, and levofloxacin was 100%. The rate of antimicrobial resistance to erythromycin and clindamycin were 98.3% and 96.6%, respectively. The dominant emm types were emm12 (65.32%), emm1 (27.61%), emm75 (2.69%), and emm89 (1.35%). Of the 297 isolates, 290 (97.64%) carried the ermB gene, and 5 (1.68%) carried the mefA gene, while none carried the ermA gene. The most common superantigen genes identified from GAS isolates were smeZ (96.97%) , speC (92.59%), speG (91.58%), and ssa (85.52%), speI (54.55%), speH (52.19%), and speA (34.34%). Isolates with the genotype emm 1 possessed speA, speC, speG, speJ, speM, ssa, and smeZ, while emm 12 possessed speC, speG, speH, speI, of mefA emm emm superantigens of GAS. The long-term monitoring of the emm type and superantigen gene analysis of GAS is crucial for understanding the variations in the GAS M protein antigen, generation of new bacterial type, epidemiological research, and vaccine preparation.


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 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 drug resistance rate to macrolides, used as alternative antibiotics, is gradually increasing, which might be related to the overuse of such antibiotics [7,8]. Our previous study has shown high frequency of resistance to erythromycin, clindamycin, and tetracycline in GAS [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 often used to track the outbreak and routinely monitor the 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 correlation between GAS diseases and emm types, superantigen gene, as well as 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 pumping mechanism mediated by mefA; the primary mechanism of resistance of various epidemic strains is different [13].
The superantigen gene is the main virulence factor and closely related to the pathogenicity of GAS.
Hitherto, 11 superantigen genes have been found, including speA, speC, speG, speH, speI, speJ, speK, speL, speM, smeZ, and ssa [14,15]. Previous investigation on the emm typing and superantigen of GAS strains from different regions of mainland China indicated that the typing and antimicrobial resistance were slightly different [16,17]. In this study, we isolated the GAS strains from patients with pharyngitis/scarlet fever and other GAS related diseases 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.

Strain collection
A total of 297 cases of GAS strains in this study were recovered from pediatric patients presenting with scarlet fever in the Children's Hospital, Capital Institute of Pediatrics, from January 2016 to December 2017. Throat swabs were obtained from patients by two physicians for routine microbiologic analysis.

Bacterial identification
The throat swab samples were inoculated on a Colombian blood plate (BD, USA) and cultured in a CO 2 incubator at 37 °C for 24-36 h. A single round colony with the transparent hemolytic ring was selected, cultured and evaluated by Gram staining. The Streptococcus typing reagents (Oxoid, Basingstoke, UK) were used to classify the suspicious colonies.

Antimicrobial susceptibility testing
According to the distribution of common antibiotics and recommendation of Clinical and Laboratory Standards Institute (CLSI), paper diffusion method (K-B method) was used to test the sensitivity of ten antibiotics to the isolated Streptococcus pyogenes. The distance between each piece of paper was > 24 mm, and the distance between the center of the paper and edge of the dish was > 15 mm. The drug-sensitive paper was placed in the plate and incubated in a 37 °C incubator. Only two plates were stacked together. After incubation for 18-24 h, the diameter of the bacteriostatic ring was measured with a Vernier caliper. The edge of the bacteriostatic ring was limited, as the bacteria cannot be observed by the naked eye. The sensitivity of bacteria was determined by the diameter of the bacteriostatic ring and CLSI standard.

DNA extraction of GAS genome
According to the recommended method on the CDC (Center for Disease Control and Prevention) website (http://www.cdc.gov/ncidod/biotechnology/stream/protocol_emm-type.htm), one ring of GAS was suspended in 300 μL saline and incubated in the water at 70 °C for 15 min. After centrifugation, the precipitate was mixed with 50 μL TE (pH 8.0), 10 μL mutanolysin (3000 U/mL), and 2 μL hyaluronidase (30 mg/mL) in a water bath at 37 °C for 30 min, boiled at 100 °C for 10 min, and centrifuged to obtain genomic DNA.

Emm genotyping
According to the recommendation of the CDC (USA), the genomic DNA of the strain and emm typing primers (emmF and emmR) were used in the reaction system. The data were uploaded to the emm typing database (http://www.cdc.gov/ncidod/biotechnology/strep/strepblast.htm) for comparison.

Erythromycin-resistance gene detection
The extracted DNA was detected by mefA, ermB, and ermA. Primer sequences are listed in Table 1.

Superantigen detection
The genomic DNA extracted from emm serotype was used to amplify 11 superantigen genes (speA, speC, speG, speH, speI, speJ, speK, speL, speM, ssa, and smez) by PCR. The primers for amplification are listed in Table 2. The product comparison predicted the size of the positive fragment and detected the superantigen carried by GAS.

Antimicrobial susceptibility testing results
292/297 cases of GAS isolates were resistant to erythromycin at a rate of 98.3%. The resistance rate of clindamycin was 96.6% (287/297). Erythromycin-resistance isolates presented a cross-resistance to clindamycin at a rate of 96.3%. The resistance rate to tetracycline was 90.23% (268/297). All strains were sensitive to penicillin, ceftriaxone, cefotaxime, cefepime, vancomycin, and levofloxacin (Table   3).  Table 4.  The details of superantigen distribution are shown in Table 6.

Discussion
S. pyogenes or GAS is a major 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]. Penicillin has always been the preferred treatment for the GAS infection. Reportedly, the minimum inhibitory concentration (MIC) value of penicillin is increasing. Also, azithromycin and other macrolides have become common antibiotics s for the treatment of pharyngitis; however, the drug resistance rate of macrolides has also been increasingly gradually [7]. Of the GAS strains isolated from the pharyngeal swabs of children with pharyngitis, 15% were not sensitive to clindamycin or erythromycin, and 12% induced resistance (D test positive) [18].
Nonetheless, the antimicrobial susceptibility results showed that all GAS strains isolated in this experiment were highly sensitive to penicillin, cephalosporin, levofloxacin, and vancomycin and highly resistant to tetracycline and macrolides, with resistance rate over 90% (Table 3). From 2016 to 2017, no significant fluctuation was detected in the resistance rate of GAS strains to antibiotics (data not shown). These findings were consistent with those from previous studies [9,19,20]. S. pyogenes is highly sensitive to penicillin, and can still be used as the first option in the clinical treatment of streptococcal infections. However, it exhibits high resistance to macrolides, such as erythromycin and clindamycin, and hence, might not be appropriate to use these antibiotics, especially macrolides, as an alternative treatment for penicillin-allergic patients. Thus, careful selection of these antibiotics is imperative.
Emm genotyping of GAS showed that the distribution of emm genotypes 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%) [14,21]. 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 [22]. In Chaoyang district, Beijing, in 2011, the main GAS epidemic strain of scarlet fever in children was emm12.0 [23]. In this study, 297 GAS strains were isolated from patients with GAS infection at the  (Table 4). Eight types of emm were found in 155 strains of GAS isolated from the pharynx of children with scarlet fever, pharyngeal tonsillitis, as well as healthy children 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 carried by healthy children [24]. From 2009 to 2016, the main emm types of GAS strains were emm12 (42.9-62.2%) and emm1 (30.7-35.0%) [25,26]]. Interestingly, the proportion of emm12 and emm1 in this experiment was similar to that reported previously. These results showed that the emm genotypes of GAS isolates changed significantly in recent years as (9%), emm12 (8%), and emm6 (7%) [29]. 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 [30]. 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 (emm 89), which was first discovered to cause tonsillar pharyngitis in Beijing, China [31], and also detected in this study. Between January 2016 and May 2017, a rare emm66.0 outbreak of GAS occurred in England and Welsh [32]. Therefore, continuous monitoring of streptococcal infection is required.
GAS infection patients with penicillin allergy are commonly treated with macrolide antibiotics. In the late 1990s, the drug resistance rate of GAS isolates to erythromycin in most regions of China was < 50%. Around 2008, the drug 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%), and emm12 was the main serotype resistant to macrolides [33]. In this study, the rate of drug resistance rate to erythromycin was 98.3%, much higher than that detected in North America and some European countries (9.6-35.8%). In the mechanism of drug resistance, 290/297 strains (97.64%) GAS strains and 5 (1.68%) mefA strains were found to carry 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 strains were resistant to erythromycin, of which, 65.1% had ermB and 32.5% had mefA as the only genes resistant to macrolides, while 2.2% had both ermB and mefA [8]. The drug 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 [34]. In Portugal, the drug resistance rates of erythromycin and clindamycin were 14% (carrying the erm(B) gene) and 9% (harbouring the erm(TR) gene) in 2010-2015, respectively [29] .Thus, it could be deduced that the high resistance rate of macrolides in China was related to the distribution of emm types.
GAS superantigens, except speG, speJ, and smeZ, were encoded by chromosome, and the remaining (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 horizontal transfer. The horizontal 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 [35]. A multicenter study in China has proved that 31.1% of the strains contain speA, while 58.6% contain speC [17]. and speJ genes increased and speA decreased. The gene spectrum of superantigen is related to the type of emm, but the same emm type strains occasionally carry different superantigen genes in the two periods. Intriguingly, no significant difference was detected in the distribution of emm types and SAg gene spectrum among different disease isolates [27]. In this study, 11 superantigens, including speC, speG, and smeZ of GAS strains were detected. Emm1 harbored speA, speC, speG, speJ, speM, ssa, and smeZ, but 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 German study showed that the most common superantigen genes in GAS were speG (92.1%), speJ (50.9%), and speC (42.0%).
Simultaneously, a correlation was established between emm type or superantigen gene and clinical complications [14]. In a rare infection outbreak in multiple trauma treatment centers, emm58 type GAS produced streptococcal exotoxins, SPEB, SPEC, SPEG, SPEF, and SMEZ; it was also termed as a macrolide-and tetracycline-resistant strain [36].  [17]. In Taiwan, strains with emm1.0, emm4.0, and emm12.0 genotype are the main causes of non-invasive diseases. Only a few strains show emm1.0 genotype containing the speC and SpeH genes, and a few with emm12.0 genotype contain speJ and smeZ genes [37]. In Spain, the emm1.0 strain of S. pyogenes associated with pharyngitis consists of speA, speG, and speJ genes, but does not have speC, speH, speI, or ssa genes [38], indicating the time-and location-dependent distribution of the emm genotypes and the superantigen gene spectrum.

Conclusions
Classification of pathogenic microorganisms is a critical method for epidemiological research. In this study, all GAS strains isolated from the Children's Hospital were sensitive to penicillin, ceftriaxone, and vancomycin, and highly resistant to erythromycin and clindamycin. Nevertheless, penicillin can still be used as the first option for the treatment of streptococcal infections. Emm gene sequence typing shows that emm12 and emm1 are the most prevalent subtypes, carrying ermB gene is the mechanism underlying resistance to macrolides, and speC, speG, and smeZ are the most common superantigens of GAS. The long-term monitoring of the emm type and superantigen gene analysis of GAS is crucial for understanding the variations in the GAS M protein antigen, generation of new bacterial type, epidemiological research, and vaccine preparation. had no role in study design; collection, analysis, and interpretation of data; writing the report; or the decision to submit the report for publication.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.