Antibiotic Resistance and Molecular Biological Characteristics of Non-13-Valent-Pneumococcal Conjugate Vaccine Serogroup 15 Streptococcus pneumoniae Isolated From Children in China

Background: The isolation rate of serogroup 15 Streptococcus pneumoniae has been increasing since developing countries began administering the 13-valent pneumococcal conjugate vaccine. Methods: We detected the antibiotic resistance and molecular characteristics of 126 serogroup 15 S. pneumoniae strains isolated from children in China. Serotypes were determined via the Quellung reaction. Antibiotic resistance was tested using the E-test or disc diffusion method. Sequence types were assigned via multilocus sequence typing. Data were analyzed using WHONET 5.6 software. Results: The frequencies of S. pneumoniae serotypes 15A, 15B, 15C, and 15F were 29.37, 40.48, 28.57, and 1.59%, respectively. Continuous-monitoring data from Beijing showed that the annual isolation rates of serogroup 15 S. pneumoniae were 7.64, 7.17, 2.58, 4.35, 3.85, 7.41, and 10.53%, respectively, from 2013 to 2019. All 126 serogroup 15 strains were susceptible to vancomycin and ceftriaxone. The non-susceptibility rate to penicillin was 78.57%. All strains were resistant to erythromycin with high minimum inhibitory concentrations (MICs). The multidrug resistance rate was 78.57%. The most common clonal complexes were CC3397, CC6011, CC10088, CC9785, and ST8589. Conclusion: Serogroup 15 S. pneumoniae is common among children in China, and these strains should be continuously monitored.

PCV13 was approved in China in November 2016 and was available in the country in May 2017. Because PCV13 is expensive and has not been included in China's national immunization program, the PCV13 vaccination rate among Chinese children is low. Another 23-valent pneumococcal polysaccharide vaccine, PPV23, which protects against serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, is available in China for children aged > 2 years. The vaccination rates for both vaccines are low. Recipients are concentrated in cities and are children whose parents can afford to pay for the vaccines. Reports on the vaccination data are scarce, and the vaccine coverage is unclear.
We sought to determine the prevalence of serogroup 15 S. pneumoniae among Chinese children as well as the characteristics of these strains. Here, we report the serotype distribution, antibiotic-resistance patterns and molecular biological characteristics of 126 serogroup 15 S. pneumoniae strains collected from children in China.

Antimicrobial Susceptibility Testing
The minimum inhibitory concentrations (MICs) of penicillin, amoxicillin, ceftriaxone, imipenem, vancomycin and erythromycin for each strain were determined using E-test strips (AB Biodisk, Solna, Sweden). Disc diffusion tests (Oxoid Ltd., Basingstoke, United Kingdom) were performed to ascertain antimicrobial susceptibilities to tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol. The results were interpreted in accordance with the Clinical and Laboratory Standards Institute 2019 guidelines, and oral breakpoints (susceptible, ≤ 0.06 mg/L; intermediate, 0.12-1.0 mg/L; resistant, ≥ 2.0 mg/L) were used for penicillin (Clinical Laboratory Standards Institute [CLSI], 2019). S. pneumoniae American Type Culture Collection 49619 was used as the quality-control strain and was included in each test set to ensure accuracy of the results. Multidrug-resistant S. pneumoniae (MDRSP) was defined as S. pneumoniae isolates that were resistant to three or more kinds of antibiotics tested in this study.

Multilocus Sequence Typing
Strains were characterized using multilocus sequence typing (MLST). Chromosomal DNA was extracted from overnight cultures of S. pneumoniae strains grown on 5% trypticase soy agar (Oxoid Ltd.) using the SiMax TM Genomic DNA Extraction Kit (SBS Genetech Co., Ltd., Beijing, China) per the manufacturer's instructions. Seven housekeeping genes (aroE, gdh, gki, recP, spi, xpt, and ddl) were amplified via polymerase chain reaction from the chromosomal DNA as described previously (Enrigh and Spratt, 1998). The products were sent to BGI Company (Beijing, China) for sequencing of both strands. The resulting sequences were compared with those of all known alleles at each locus, as well as with the sequence types (STs) in the pneumococcal MLST database. 1 New alleles and allelic profiles were submitted to the MLST database for name assignment. goeBURST v1.2.1 2 was used to investigate relationships among the strains and assign strains to a clonal complex (CC) based on the stringent group definition of six of the seven shared alleles.

Statistical Analysis
Antimicrobial susceptibility and MLST data were analyzed using WHONET 5.6 as recommended by the World Health Organization.

DISCUSSION
Since the introduction of PCV13, many countries and regions that included PCV13 in their national immunization programs have reported increased infections from serogroup 15 S. pneumoniae, which is not covered by the PCV13 vaccine (Linden et al., 2015;Sheppard et al., 2016;Nakano et al., 2020). Serogroup 15 S. pneumoniae strains have caused outbreaks (Katherine et al., 2012) and deaths (Arushothy et al., 2020) among children. China has not yet included PCV13 in the national immunization program, and the PCV13 vaccination rate among Chinese children is low. Additionally, the epidemiological monitoring data for S. pneumoniae in Chinese children are inexact; thus, the prevalence of serogroup 15 S. pneumoniae and the characteristics of these strains are unclear. Here, we reported the serotype distribution, antibioticresistance patterns, and ST characteristics of 126 serogroup 15 S. pneumoniae strains isolated from children in China.
Half of these strains came from the continuous monitoring of Beijing Children's Hospital, which is one of the few continuousmonitoring data programs in mainland China and can partially reflect the prevalence of S. pneumoniae in Chinese children. During the study period, the overall isolation rate of serogroup 15 S. pneumoniae in the child population of Beijing Children's Hospital was 6.12%. After PCV13 was launched in China in May 2017, the isolation rates of serogroup 15 S. pneumoniae in 2018 and 2019 were 7.41 and 10.53%, respectively, showing an increasing trend.
The Pneumococcal Molecular Epidemiology Network (PMEN) 3 was established in 1997 for global surveillance of antibiotic-resistant S. pneumoniae and to standardize the nomenclature and classification of resistant clones. One 15A serotype was included among the 43 drug-resistant clones spread worldwide and published by the PMEN: Sweden 15A -25 (ST63). Over the past few years, several studies have reported increasing numbers of serotype 15A-ST63 strains (Ho et al., 2015;Sheppard et al., 2016;Nakano et al., 2018Nakano et al., , 2019Nakano et al., , 2020. In this study, ST63 accounted for 5.41% (2/37) of the serotype 15A strains, which was far lower than the 88.24% reported in Hong Kong (Ho et al., 2015) and the 98.28% reported in Japan (Nakano et al., 2020). Some studies FIGURE 2 | Distribution of sequence types (STs) amongst the clonal complexes of serogroup 15 Streptococcus pneumoniae strains. goeBURST analysis was performed on the multilocus sequence typing data generated from all 126 strains analyzed. STs linked by a line belong to the same cluster. reported that ST63 showed high multidrug-resistance rates (Sheppard et al., 2016;Nakano et al., 2018). Both ST63 strains in this study were resistant to erythromycin; one of these was also resistant to tetracycline, and the other was non-susceptible to imipenem, but both strains were sensitive to other antibiotics. The significant differences in isolation rates and antibiotic susceptibility characteristics of ST63 between our study and the other studies may be due to regional differences.
We found that serogroup 15 S. pneumoniae strains showed good sensitivity to common antibiotics. However, the most common CC, CC3397, was 100% non-susceptible to penicillin. We wondered whether the prevalence of CC3397 in serogroup 15 S. pneumoniae results from antibiotic pressure. Many studies have reported clonal shift phenomena in other serotypes, as CC271 replaced CC983 among serotype 19F strains , ST81 replaced ST342 amongst serotype 23F pneumococcal isolates , CC320 replaced CC230 in 19A strains (Beall et al., 2011;Golden et al., 2015;Mayanskiy et al., 2017), and CC876 replaced CC875 in serotype 14 strains (He et al., 2015). These examples of clonal shift phenomena in the same serotype may be caused by antibiotic pressure, as they followed the principle that CCs/STs expressing high antibiotic resistance will replace CCs/STs with lower antibiotic resistance. Presently, the epidemiological characteristics of serogroup 15 S. pneumoniae are not being continuously monitored to access the changes in CC/ST prevalence in China. Whether CC3397 is the most drug-resistant of the serogroup 15 S. pneumoniae clones or whether CC3397 will be replaced by more drug-resistant clones in the future is unclear. Therefore, clinicians must strengthen surveillance of the antibiotic resistance and other epidemiological characteristics of serogroup 15 S. pneumoniae.

CONCLUSION
Serogroup 15 S. pneumoniae is common among children in China. Because CC3397, the main CC of serogroup 15 S. pneumoniae, showed high non-susceptibility to penicillin, long-term monitoring of antibiotic susceptibility and other epidemiological characteristics of serogroup 15 S. pneumoniae is essential.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
WS, QD, LY, and WG conducted the experiments. WS and QW were responsible for the laboratory analysis. WS and KY designed the study, collected the data, analyzed the data, interpreted the results, and drafted the manuscript. All authors reviewed and revised the manuscript and approved the final version.

FUNDING
This study was supported by the Beijing Natural Science Foundation (L202004).