Antibiotic susceptibility and clonal distribution of Staphylococcus aureus from pediatric skin and soft tissue infections: 10-year trends in multicenter investigation in China

Background Skin and Soft Tissue Infections (SSTIs) Surveillance Network of S. aureus In Pediatrics in China was established in 2009 to routinely report epidemiological changes. We aimed to monitor the present antibiotic sensitivity and molecular characteristics of S. aureus and methicillin-resistant S. aureus (MRSA) from SSTIs in children nationwide and track the changes over the past decade. Methods Patients diagnosed with SSTIs from the dermatology departments of 22 tertiary pediatric hospitals in seven geographical regions of China were recruited continuously from May 2019 to August 2021. S. aureus was isolated, and its sensitivity to 15 antimicrobials was evaluated using the broth microdilution method. The molecular characteristics of the MRSA isolates were determined through multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. The presence of the Panton–Valentine leukocidin gene (pvl) was determined. Results The detection rate of S. aureus was 62.57% (1379/2204), among which MRSA accounted for 14.79% (204/1379), significantly higher than the result in previous study in 2009-2011 (2.58%, 44/1075). Compared with previous study, the sensitivity to cephalosporins and fusidic acid decreased to varying degrees, while that to chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, penicillin, and tetracycline increased significantly. The sensitivity to mupirocin, trimethoprim/sulfamethoxazole (TRISUL), and rifampicin still maintained at a high level (97.90%, 99.35% and 96.66% respectively). The leading multidrug resistance pattern of MRSA and methicillin-sensitive S. aureus (MSSA) were erythromycin-clindamycin-tetracycline (55.84%; 43/77) and erythromycin-clindamycin-chloramphenicol (27.85%, 44/158) respectively. 12 high-level mupirocin-resistant strains were detected, and notable differences in geographical distribution and seasonal variation were observed. The main types of MRSA were ST121 (46.08%, 94/204), followed by ST59 (19.61%, 40/204). SCCmec V (65.69%, 134/204) and SCCmec IV (31.86%, 65/204) were dominant epidemic types. ST121-V, ST59-IV, and ST22-V were the most prevalent clones nationwide. The detection rate of pvl had increased markedly from 9.09% (4/44) in 2009-2011 to 22.55% (46/204) in 2019-2021 (P<0.05). Conclusion The antibiotic sensitivity and molecular characteristics of S. aureus from pediatric SSTIs has changed significantly over the past decade. To standardize medical care, provide timely and reasonable clinical treatment, and effectively manage infection control, Chinese pediatric SSTIs guidelines are urgently needed.


Introduction
Staphylococcus aureus is the main pathogen that causes skin and soft tissue infections (SSTIs), such as pustules, folliculitis, boils, and abscesses, in pediatric patients (Lowy, 1998) as well as fatal infections, such as necrotizing fasciitis and toxic shock syndrome (Burnham and Kollef, 2018). Methicillin-resistant S. aureus (MRSA) has attracted considerable attention owing to its drug resistance and virulence (Lee et al., 2018). Community-associated MRSA (CA-MRSA) mainly causes SSTIs in young and healthy people in the community (Turner et al., 2019). Over the past decade, CA-MRSA has been considered to be the main cause of the increased burden of MRSA diseases. Some CA-MRSA strains have been increasingly involved in nosocomial infections and have even become dominant strains in medical settings (Elston and Barlow, 2009). The resistance rate of CA-MRSA is increasing, not only to b-lactam antibiotics but also to non-b-lactam antibiotics (Guo et al., 2020). The spectrum of CA-MRSA invasive diseases is expanding and is increasingly becoming the focus of global infection (Hassoun et al., 2017). Epidemiological information on MRSA is important for clinical decision-making and public health monitoring. Furthermore, classification of MRSA is an important part of describing epidemiological trends and formulating infection control strategies (Mediavilla et al., 2012).
To our knowledge, few studies on the antibiotic sensitivity of S. aureus and molecular characteristics of MRSA from SSTIs in China have been conducted (Xiao et al., 2019;Zhao et al., 2022). Furthermore, results from different regions differ, and national studies related to children are rare. A national SSTIs surveillance network of S. aureus in pediatrics, established in 2009 and led by the Department of Dermatology, Beijing Children's Hospital, is the only available nationwide epidemiological surveillance network with regular investigation in China (Liu et al., 2016). Currently, 22 children's hospitals have joined. The present study aimed to track the changes in antibiotic sensitivity of S. aureus as well as the molecular characteristics and epidemiology of MRSA in children diagnosed with SSTIs in China.

Patient enrollment
This was a multi-center, cross-sectional epidemiological study on children diagnosed with SSTIs in the dermatology departments of 22 tertiary pediatric hospitals. According to their geographical location, the hospitals were divided into seven groups: North, Middle, East, South, Northeast, Northwest, and Southwest China. All patients who met the criteria for SSTIs were recruited continuously from the dermatology departments from May 2019 to August 2021. The inclusion criteria were as follows: 1) no history of major congenital malformations or severe chronic diseases, 2) no history of surgery or hospitalization within the past year, 3) no history of dialysis or deep catheterization, and 4) no history of antibiotic use within the past week. Information, including sex, age, predisposing factors, disease type, specimen collection time, and basic medical history, was collected. Patients who could not provide general information were excluded. The enrolled patients were treated according to routine treatment, and the swab specimens of infection sites from the non-repetitive participants were collected continuously.

Strain identification
The isolated strains were identified using traditional microbial identification methods, coagulase and catalase tests, and latex slide agglutination test (Oxoid Ltd., Basingstoke, UK). All three tests were positive for S. aureus (Weist et al., 2006).

Identification of MRSA strains
In addition to resistance to cefoxitin and oxacillin, MRSA was further identified through polymerase chain reaction (PCR) amplification of the nuc and mecA genes according to the method described previously (Sahebnasagh et al., 2014). ATCC 29213 and ATCC35601 were used as a negative and positive control, respectively, for the mecA gene.

Susceptibility profiles
The antibiotic susceptibility profiles of the S. aureus isolates were determined using the Sensititre ® Antimicrobial Susceptibility Testing System (Thermo Scientific, UK), following the manufacturer's instructions. The minimum inhibitory concentration (MIC) to 15 antibiotics (cefazolin, ceftriaxone, cefuroxime, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, fusidic acid, gentamicin, mupirocin, penicillin, rifampicin, tetracycline, TRISUL, and vancomycin) were detected using the broth microdilution method (Novy et al., 2014). S. aureus ATCC 29213 and ATCC 35601 were used for quality control. The antimicrobial sensitivity breakpoints were interpreted according to the current Clinical and Laboratory Standards Institute (CLSI) breakpoints for S. aureus (CLSI, 2019), while sensitivity to cefazolin, ceftriaxone, and cefuroxime was interpreted according to a previous version of CLSI (Clinical and Laboratory Standards Institute/NCCLS, 2005). An E-test ((bioMeŕieux, Marcy-L'Etoile, France) was further performed on the isolates classified as mupirocin-resistant through broth microdilution.
Epidemiological typing of MRSA DNA was extracted for PCR using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology, China). Multilocus sequence typing (MLST) was performed on MRSA isolates using the method described previously (Enright et al., 2000). Sequences of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) were compared with known alleles from the MLST database (https:// pubmlst.org/organisms/staphylococcus-aureus). Allelic profiles and sequence types (STs) were determined using the database.
The isolates were also subjected to staphylococcal cassette chromosome mec (SCCmec) typing, which is based on multiplex PCR with 10 primers (Omuse et al., 2016). SCCmec types I-V were assigned according to the combination of the cassette chromosome recombinase (ccr) type and mec class. Isolates that could not be assigned to any expected type were defined as non-typable.

Panton-Valentine leukocidin gene detection
pvl was amplified using PCR as described previously (Hesje et al., 2011) with minor modifications. MRSA N315 was used as a negative control, while ATCC 25923 was used as a positive control.

Statistical analysis
A database including the age, sex and disease patterns of the patients, antimicrobial resistance and molecular characteristics of the corresponding isolate was constructed in Microsoft Excel 2003. GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, United States) was used to create graphs. All susceptibility data were analyzed using WHONET software (version 5.6). JMP ® 11 Statistical Discovery Software (S.A.S. Institute Inc., Cary, North Carolina) was used for statistical analysis. Categorical variables were analyzed using the chisquared (c2) or Fisher's exact test. Significance was set at P < 0.05.

General information
The initial data of S. aureus and MRSA collection as well as the distribution of them are summarized here. The clinical features of patients from whom the strains were collected are analyzed as follows. The age of patients ranged from 3 days to 18 years old, with 77.16% (1064/1379) aged 1-6 years. Of the patients, 58.45% (806/1379) were males and 41.55% (573/1379) were females. Detailed clinical features are presented in Table 1.

Characteristics of resistance pattern of S. aureus and change trend
The results of the antimicrobial susceptibility test on strains collected in this present study are shown in Table 2. The resistance The distribution of specimens, S. aureus and MRSA as well as prevalent clones, by region. All seven geographical regions were included as follows: Northeast, green, North China, gray; East China, blue; Northwest, yellow; Middle China, khaiki; Southwest, pink; South China, orange. *pvl-positive clones contained. rates of MRSA to ciprofloxacin, clindamycin, erythromycin, and tetracycline were significantly higher than those of MSSA (P<0.05). S. aureus with resistance to three or more classes of antimicrobial agents were defined as multidrug-resistant (MDR). In this study, MDR was observed in 37.75% (77/204) of MRSA strains and 13.45% (158/ 1175) of MSSA strains. The predominant resistance patterns of MRSA to non-b-lactam antibiotics were erythromycin-clindamycin-tetracycline (55.84%; 43/77), followed by erythromycin-clindamycin-tetracyclinechloramphenicol (18.18%; 14/77). The resistance patterns of MSSA were very different from that of MRSA, the most common profiles of which were erythromycin-clindamycin-chloramphenicol (27.85%, 44/ 158) and erythromycin-clindamycin-tetracycline (22.15%, 35/158). Among different STs of MRSA strains, the proportion of MDR in ST121 was the highest (49.35%; 38/77), followed by ST59 (22.08%; 17/ 77) and ST338 (6.49%; 5/77).
The changes of resistance patterns of S. aureus collected in this study with strains collected in 2009-2011were analyzed. The current resistance rates of S. aureus to cefazolin, ceftriaxone, cefuroxime, and fusidic acid had increased significantly (P<0.05), and that to chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, rifampicin, tetracycline, and TRISUL decreased significantly (P<0.0001), while no significant difference was found in resistance to vancomycin and mupirocin. Besides, the resistance rate to penicillin decreased from 96.8% to 94.13% (P=0.0004). The comparison of the antimicrobial sensitivity of S. aureus isolates between 2009-2011 and 2019-2021 is shown in Figure 3.
Major features of ST121 and ST59 strains ST121 was the most prevalent type of MRSA strains, which all typed as SCCmec V and pvl negative. ST121 strains were mainly distributed in Northwest and South China with a positive rate of 80.00% (4/5) and 69.05% (58/84), respectively, while they were not detected in Northeast China. Compared with non-ST121 isolates, ST121 isolates had a significantly higher resistance rate to clindamycin and lower resistance rates to cefazolin, cefuroxime, and ciprofloxacin. The detection rate of ST59 was second to that of ST121. ST59, which was opposite to ST121, was mainly distributed in Northeast and North China, with a positive rate of 80.00% (16/ 20) and 40.91% (9/22), respectively. Inconsistent with ST121, ST59 strains were mainly typed as SCCmec IV (77.50%, 31/40). The positive rate of pvl was 35.00% (14/40), significantly higher than that of ST121 (0.00%, 0/94). The resistance rate of ST59 isolates to ciprofloxacin, cefazolin, and cefuroxime was significantly higher than that of ST121 isolates (P<0.05), and there was no significant difference in the resistance rate to other antibiotics.

Discussion
This study was the second to conduct national multicenter epidemiological monitoring of S. aureus for SSTIs in pediatrics since the surveillance network was established in China in 2009. The epidemiological trends of SSTIs in 22 tertiary children's hospitals in seven geographical regions of mainland China were investigated. Compared with the study conducted 10 years ago, we presented four major findings: 1) the resistance profiles of S. aureus isolates had changed considerably; 2) the prevalence of CA-MRSA and its pvl-positive strains, increased significantly; 3) the proportion of deep infections increased significantly; and 4) ST121-V was the dominant clone, with the percentage increased.
In this study, MRSA accounted for 14.79%, significantly higher than in 2009-2011 (2.58%). It was reported that antimicrobial agents should be chosen to target MRSA and MSSA if MRSA accounts for >10% of S. aureus among SSTIs (David and Daum, 2017). Either clindamycin or TRISUL was recommended because of the low cost and activity against community-associated MRSA and MSSA strains of each of these drugs (Williams et al., 2011). According to previous studies, one of these antimicrobials should be used in addition to incision and drainage for a skin infection (Miller et al., 2015;Daum et al., 2017). In this study, we identified a low resistance rate of S. aureus to TRISUL (0.4%-2%). The longterm use of TRISUL remains a suitable option for treating complex hyperimmunoglobulin E syndrome and chronic granulomatous disease (Hashemi et al., 2017). Clindamycin was reported to be effective at treating infections caused by susceptible CA-MRSA isolates (Miller et al., 2015). However, in this study, though significantly decreased compared with that in 2009-2011, the resistance rate of clindamycin was still higher than 50%, indicating that it is not a good choice for treating SSTIs in children in China.
For empiric or targeted therapy for S. aureus, an antistaphylococcal b-lactam drug was the most appropriate choice (David and Daum, 2010). Furthermore, it was reported that cephalosporins and penicillin are most commonly used in China (Li et al., 2016). Therefore, the MICs of the clinical strains to penicillin as well as cephalosporins was detected to track changes in sensitivity. The resistance rates of S. aureus to penicillin, though decreased significantly, remained at a high level (94.13%). On the contrary, the sensitivity to cephalosporins was maintained at a high level (90.65%-97.53%). According to this result, cephalosporins might be a suitable alternative to penicillin for empiric therapy.MDR to non-b-lactam antibiotics was detected in both MRSA and MSSA. The presence of MDR strains in outpatients with SSTIs can lead to persistent or recurrent MRSA infections (Lee et al., 2017).
Clinically, most SSTIs can be controlled only with topical antibiotics. Mupirocin is a topical antibiotic that has been extensively used for treating MRSA skin and soft-tissue infections, decreasing certain types of surgical site infections and eliminating nasal colonisation of MRSA among patients and medical staff. In the present study, the sensitivity to mupirocin was still high,    consistent with previous results (Liu et al., 2016;Chen et al., 2020). However, high-level mupirocin strains were detected in both MRSA and MSSA strains in this study. Dadashi et al. reported that the incidence of high-level mupirocin resistance in MRSA was the highest in Asia (12.1%), followed by Europe (8.0%) and the USA (5.9%) (Dadashi et al., 2020). In China, it was reported that mupA mainly accounted for high level mupirocin resistance (Jin et al., 2018;Guo et al., 2023). The mupA gene is typically located on mobile genetic elements and is plasmid mediated, which maybe the reasons for transmission of clones (Liu et al., 2010). In this study, high-level mupirocin resistant strains were mainly isolated from South China, which were generally with higher economical levels than others. Easy access to antibiotics without prescriptions, a high rate of antibiotic misuse, and the frequency of empiric treatment in these regions may lead to the situation. The results suggested that the rational use of mupirocin should be strengthened, and drug resistance should be further monitored. Fusidic acid was also an important choice for SSTIs. The worldwide resistance rate of S. aureus, especially MRSA, to fusidic acid was reported to be 0.3%-64% (Gajdaćs, 2019). In the present study, the resistance rate of S. aureus to fusidic acid increased from 1.8% to 4.57%, which was still low, similar to that identified previously (Gu et al., 2016). The resistance rate in MSSA (4.85%) was higher than that in MRSA (2.94%), consistent with previous study (Zhanel et al., 2021). The increased detection rate of fusidic acid-resistant strains suggests that the drug should be used in moderation.
Based on the above results of antimicrobial sensitivity, we call for the timely introduction of guidelines for the treatment of SSTIs in children in China to develop scientific and effective diagnosis and treatment programs.
MRSA has been the focus of global SSTIs (Mediavilla et al., 2012). Recently, an upward trend in the incidence of pvl-positive MRSA was observed in Europe and Japan (Bouchiat et al., 2017;Nakaminami et al., 2020). Concern was raised as pvl-positive MRSA strains usually cause deep infections such as furuncles and abscesses (Shallcross et al., 2013). Compared with the study conducted 10 years ago, the detection rate of MRSA in the present study had increased by 5.65-fold (14.79% vs. 2.58%; P<0.0001), and the positive rate of pvl had increased by 2.3-fold (22.55% vs. 9.8%; P<0.05). In addition, the infection spectrum had changed, as deep infections including folliculitis, furuncle, and abscesses increased significantly, while superficial infections decreased. Therefore, according to our surveillance, there was an increasing trend in the prevalence of pvl-positive MRSA among SSTI isolates in Chinese children, which was probably connected with the increase in deep infections. Attention should be paid to the surveillance of pvlpositive MRSA in the future.
MLST is a universal method for understanding the molecular epidemiology of MRSA (Enright et al., 2000). Previous studies demonstrated that the most prevalent clones of CA-MRSA from SSTIs had unique geographic distribution, as ST8 was mostly reported in the USA (Otter and French, 2010), while ST80 was mainly in Europe, and ST59 was mainly in the Asia-Pacific region (Deurenberg and Stobberingh, 2008). In mainland of China, Taiwan, and Hong Kong, ST59 was reported as the most prevalent ST of MRSA strains from SSTIs (Yu et al., 2015), while ST121 was rarely dominant for clinical infections (Chuang and Huang, 2013;Wang et al., 2019). The epidemiological data hint that most ST121 strains were MSSA (Goering et al., 2008;Rao et al., 2015). However, in the present survey, ST121 (46.08%; 94/204) was the dominant ST in MRSA strains, followed by ST59, which was consistent with the results of our previous study (Liu et al., 2016). This was probably due to the differences in the population. In the present study, the enrolled children were at preschool age (1-6 years). It was reported that among preschool children, ST121 was the most prevalent clone in China (Fan et al., 2009). Besides, they were all outpatients who had no history of hospitalization. Therefore, the study was more representative of infections from the community.
In conclusion, tremendous changes in the antibiotic sensitivity of S. aureus from SSTIs in Chinese children had been observed compared with the results obtained 10 years ago. The incidence of MRSA as well as the positive rate of pvl had increased significantly, with ST121, ST59, and ST22 being the main epidemic types. With the significant changes, further research tracking sensitivity to antibiotics as well as the molecular epidemiological characteristics of MRSA is needed. Moreover, to standardize medical care, help clinicians make evidence-based treatment decisions, and effectively manage infection control, guidelines for SSTIs in pediatrics in China are urgently needed.

Data availability statement
The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.

Ethics statement
This project (2019-k-123) was approved by the Research Ethics Committee in Beijing Children's Hospital, China on May 21, 2019.

Author contributions
YL, KY, YY and LM designed the study. WS, YL, QW, LY and WG conducted the experiments. WS, YL and QW collected and analyzed the data, interpreted the results, and drafted the manuscript. All authors contributed to the article and approved the submitted version.