Molecular characterization of invasive Streptococcus pneumoniae clinical isolates from a tertiary children’s hospital in eastern China

ABSTRACT Streptococcus pneumoniae is a common opportunistic pathogen that causes invasive pneumococcal disease (IPD), especially in children. This study aimed to determine the prevalence and molecular characteristics of S. pneumoniae isolated from children with IPD. A total of 78 S. pneumoniae isolates from aseptic body fluids of 70 IPD patients were collected at the Children’s Hospital of Nanjing Medical University (Jiangsu Province, China) during 2017–2021. Whole-genome sequencing technology was used to analyze the serotype, sequence type (ST), virulence, and antibiotic resistance of the 78 invasive S. pneumoniae clinical isolates. Our results showed that the pneumococcal infection rate declined after the COVID-19 outbreak in 2019. Serotypes 19F, 14, 6A, 23F, 19A, and 6B were the most common strains. The pneumococcal conjugate vaccine (PCV) 13 serotype coverage rate was 87.1%. All isolates were classified by multi-locus sequence typing (MLST) analysis into 27 different STs, including 3 novel STs (ST17941, ST17942, and ST17944) and 1 novel allele [recP (558)]. The most predominant ST was ST271, followed by ST320 and ST876. All isolates carried the following virulence genes: cbpG, lytB, lytC, pce (cbpE), pavA, slrA, plr (gapA), hysA, nanA, eno, piuA, psaA, cppA, iga, htrA (degP), tig (ropA), zmpB, and ply. All isolates were multidrug resistant and had high levels of resistance to macrolides, tetracyclines, and sulfonamides. Taken together, this study revealed extensive genetic diversity among S. pneumoniae isolates from a single Chinese hospital. Wearing masks, universal infant vaccination with PCV13, and the launch of recombinant protein vaccine development programs could reduce the burden of IPD in children. IMPORTANCE Invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae in children remains a global burden and should be given more attention due to the fact that the pneumococcal vaccine is not fully covered globally. The molecular epidemiological characteristics of S. pneumoniae are not so clear, especially in these years of COVID-19. In this study, we collected S. pneumoniae isolates from the aseptic body fluid of children with IPD from 2017 to 2021 in a tertiary children’s hospital in China and revealed the extensive genetic diversity of these isolates. Most importantly, we first found that the rate of pneumococcal infection has declined since the COVID-19 outbreak in 2019, which means that wearing masks could reduce the transmission of S. pneumoniae. In addition, it was shown that universal infant vaccination with PCV13 seems essential for reducing the burden of IPD in children.

to respiratory tract infections but also to invasive pneumococcal disease (IPD), such as sepsis, meningitis, and pleurisy (2).IPDs are characterized by high morbidity and mortality rates worldwide and mainly affect children <5 years old, particularly in developing countries.The Global Burden of Diseases, Injuries, and Risk Factors Study 2017 estimated that approximately 380,900 children <5 years old die from pneumococ cal disease.In the same year, S. pneumoniae was listed as 1 of the 12 priority pathogens by the World Health Organization (1).
There are at least 100 S. pneumoniae serotypes circulating worldwide.The biochemi cal structure of the capsular polysaccharides (CPS) serves as the basis for categorizing the S. pneumoniae serotypes (3).The CPS, as a virulence element affecting nearly every link of pneumococcal pathogenesis, are considered a prerequisite for IPD (4).Pneumococcal vaccines are designed and developed based on conjugated polysaccharides and are a remarkably effective means of preventing pneumococcal conjugate vaccine (PCV)-sero type pneumococcal disease.Indeed, timely tracing of the epidemiology of S. pneumoniae in children could contribute to the development of pneumococcal vaccine coverage.
S. pneumoniae virulence is attributed to CPS and multiple factors that mediate inflammatory responses via proteases serving dual roles (S. pneumoniae self-protein processors and host protein targets) (5).Virulence factors with unique biological functions mediate the adhesiveness and invasiveness of S. pneumoniae, as well as the pathogenesis underlying IPD (1).Virulence factors participate in inflammatory reactions at different infection sites, such as ClpP, BacB, and HtrA in the lung; CbpG, DacB, and ZmpC in the blood; and CbpA, ChoP, and PAFr at the blood-brain barrier (5,6).Moreover, virulence factors have an impact on disease severity and the pathogenicity of the host (6).Therefore, monitoring the virulence profile is crucial and not only helps clinicians understand disease severity at an early stage but also facilitates the implementation of empirically targeted therapy.
The primary consideration in the clinical treatment of IPD is antimicrobial therapy.S. pneumoniae resistance to commonly used antibiotics, such as penicillins, macrolides, cephalosporins, and sulfonamides, has become a challenging problem, especially in Asian countries (7).Although large-scale pneumococcal vaccination has been intro duced, S. pneumoniae drug resistance is still a very serious issue in countries where antibiotics are widely used, drug-resistant clones are widespread, and vaccines are less frequently administered.Therefore, it is essential to regulate antimicrobial resistance based on its regional status.
In the current study, whole-genome sequencing (WGS) was used to determine the prevalence and molecular characteristics of invasive S. pneumoniae strains isolated from patients admitted to the Children's Hospital of Nanjing Medical University (Jiangsu Province, China) between 2017 and 2021.The aim of this study was to provide a vaccination and antibiotic therapy reference for the prevention and control of IPD in children.

Bacterial isolates and clinical information
This study was conducted at the Children's Hospital of Nanjing Medical University (Jiangsu Province, China) between 2017 and 2021.The Children's Hospital of Nanjing Medical University is one of the largest pediatric hospitals in Jiangsu Province, with 1,742 beds and approximately 73,600 hospital admissions per year.
S. pneumoniae isolates were collected in this study when clinicians defined the isolates as infectious pathogens.Aseptic body fluids (e.g., cerebrospinal, blood, and pleural fluids) were collected from children infected with S. pneumoniae during hospitalization.Cultures were isolated on Columbia blood agar plates (Kemaja, Shanghai, China), which were incubated for 24-48 h in 5% carbon dioxide at 35°C.All isolates were identified as S. pneumoniae using a VITEK-2 compact system (bioMérieux, Marcy-l'Étoile, France) and optochin susceptibility testing.
The electronic case records, including pediatric patient demographics, specimen information, infection characteristics, antimicrobial treatment, and clinical outcome, were retrieved and reviewed.
The study was conducted in accordance with the Declaration of Helsinki and approved by the Clinical Trial Ethics Committee of the Children's Hospital of Nanjing Medical University (No. 202301027-1).

Serotyping
The S. pneumoniae serotypes were analyzed using Pneumococcal Capsular Typing (PneumoCaT, version 1.2.1; https://github.com/phe-bioinformatics/PneumoCaT),which used a two-step approach to assign capsular type to S. pneumoniae genomic data (9).In the first step, if the readset matched >90% to a single capsular locus sequence, the capsular type was reported; otherwise, the next step would commence.Serotypes within a serogroup/genogroup were distinguished by utilizing the capsular-type variant database.

Multi-locus sequence typing
The sequence types (STs) of S. pneumoniae were analyzed utilizing multi-locus sequence typing (MLST) software (version 2.22.1, tseemann; https://github.com/tseemann/mlst).New alleles were amplified by polymerase chain reaction and sequenced using the Sanger method (10).Obtained sequences and strain-related information were submitted to the pneumococcal PubMLST database to automatically assign a new ST accession number.

Statistical analysis
The Statistical Package for Social Sciences software (version 22.0; SPSS, Chicago, IL, USA) was used to analyze the statistical significance of the data.A chi-square test was used to determine whether the differences among the two groups were statistically significant.The Bonferroni method based on the chi-square test was used to determine whether the differences among multiple groups were statistically significant (16).The Bonferroni method compares a set of n tests in pairs and uses P < 0.05/n as the judgment criterion for correction (18).

Clinical characteristics
A total of 70 IPD cases were collected from the Children's Hospital of Nanjing Medical University between 2017 and 2021.The strains isolated from eight children with double infections (bloodstream and cerebrospinal fluid) were enrolled in this study.Finally, we collected 78 S. pneumoniae isolates to conduct the study.The incidence of the cases in the past 5 years had a mountain-like profile: 2019 was the peak; the incidence of the cases had an upward trend before 2019; and the incidence of the cases had a downward trend after 2019.The demographic and clinical characteristics of the pediatric patients are shown in Table 1.

MLST
Given that the isolates from the two specimen sources of each pediatric patient had the same ST, we only included one isolate in the statistical analysis.All isolates were classified by MLST analysis into 27 different STs, including 3 novel STs (ST17941, ST17942, and ST17944) and 1 novel allele [recP ( 558 By comparing the strains with the pneumococcal molecular epidemiology network clones, two global clones with at least six of seven MLST alleles shared were identified in this study, including Spain23F-1 [n = 3 (4.2%)]and Taiwan19F-14 [n = 1 (1.4%)].Two clonal complexes (CCs) and 19 singletons were obtained via the goeBURST algorithm (goeBURST distance) at SLV level 1 (Fig. 2A).CC271 (including ST271, ST1937, ST236, ST1453, and ST320) was the most prevalent CC, which accounted for 40.0%(n = 28/70) of the strains, followed by CC876 (including ST876, ST17941, and ST17942), which accoun ted for 12.9% (n = 9/70) of the strains.
The epidemiologic trends of MLST with serotype, year, and outcome were analyzed via the goeBURST Full MST algorithm (goeBURST distance) at LV level 6.Strains of the same serotype showed an aggregation trend in which ST271, ST320, ST876, ST90, and ST902 were associated with serotypes 19F, 19A, 14, 6B, and 06A, respectively (Fig. 2B).Moreover, the same ST-type strains mostly showed an annual aggregation distribution (Fig. 2C).Notably, most ST-type strains had a good outcome, but strains with a poor outcome tended to be concentrated in prevalent strains, such as ST271, ST320, and ST876 (Fig. 2D).
Among the strains of different CCs, the related gene-carrying rates varied, as shown in Table 2.For adherence factors in particular, the pspC/cbpA carriage rate was highest in CC271 and lowest in CC876 (93.5% vs 11.1%; P = 0.000).

Relationship between antibiotic susceptibility and molecular characteristics
The antibiotic resistance phenotypes and genes of 78 S. pneumoniae isolates are displayed in Table 3.As shown in Table 3, all of the strains isolated from patients with meningitis were resistant to PEN, while the strains isolated from non-meningitis patients were susceptible to PEN.The CRO and CTX resistance rates of the strains isolated from meningitis patients were significantly higher than the strains isolated from non-meningi tis patients (16.7% vs 14.8% and 41.7% vs 18.5%, respectively; P = 0.000).Most isolates showed high resistance to ERY (100.0%) and TET (93.6%), and a considerable number of isolates were resistant to SXT (69.2%).None of the strains were resistant to LZD or VAN.All of the isolates exhibiting resistance to at least one antimicrobial drug in three or more antimicrobial categories were defined as having multidrug resistance (MDR).Notably, there were differences in the drug sensitivity results of the strains isolated from different specimen sources for each pediatric patient.
The SXT resistance rates of the serotype 19F strains were significantly higher than the other serotype strains, accounting for 95.8%.The antibiotic resistance phenotype varied by two CCs.For example, the TET and SXT resistance rates of the CC271 strains were markedly higher than the CC876 strains (96.8% vs 55.6%; P = 0.001 and 96.8% vs 0%; P = 0.000).

DISCUSSION
Pediatric IPD remains a global burden, although the pneumococcal disease mortality rate in children has declined substantially in the pneumococcal vaccine era (19).Pediatric IPD The characteristics of virulence genes in 78 strains with different serotypes and CCs.The Bonferroni method based on the chi-square test was used to test whether the differences among multiple groups were statistically significant.According to the Bonferroni method, when P < 0.0166, the differences among three groups were statistically significant; when P < 0.0125, the differences among four groups were statistically significant."Bold" means that the difference is statistically significant, and "N.A. " means no difference among multiple groups."*" indicates P < 0.05."#" indicates the situation where P < 0.05 but does not match the chi-square test with the Bonferroni method.The symbols (α, β,γ) represent the groups with significant differences.Compare multiple sets of data in pairs based on the first group in the Bonferroni method: P < 0.05/n, "γ" means significant differences; P = 0.05/n, "β" means unable to determine; P > 0.05/n, "α" means no significant differences.
should be given more attention because the pneumococcal vaccine is not administered universally in China.Indeed, the Children's Hospital of Nanjing Medical University is the only children's medical center in Jiangsu Province that receives nearly all pediatric patients throughout the province.Thus, this study accurately reflects the epidemiology of IPD in children within the entire Jiangsu Province.In the current study, the pneumo coccal infection rate declined after the COVID-19 outbreak in 2019, which indicates that wearing masks prevents S. pneumoniae droplet transmission and reduces the infection rate.In fact, a recent survey showed that S. pneumoniae was the most common coinfected bacteria among COVID-19 patients in Jiangsu Province in 2020 (20).Therefore, it is essential to conduct an epidemiologic investigation of IPD in children.
The distribution of serotypes varies with geographic region, age, infection type, and time.In this study, the most prevalent distribution of serotypes among invasive S. pneumoniae-infected children in Nanjing was 19F, 14, 6A, 23F, 19A, and 6B, which were similar to other geographic regions of China, such as southwest China (19F, 19A, 6B, 6A, and 14), East China (19F, 6A/B, 23F, 14, and 15A), Shanghai (19F, 19A, 6A, 14, and 6B), and Beijing (19F, 19A, 23F, 14, and 6A) (15,16,21,22).Conversely, there were differences in the serotype distribution in international regions, such as Africa (28F, 6A, 11A, 3, and 7C) and Egypt (1, 6ABC, 19F, 5, and 18ABC) (3,23,24).The serotype distribution discrepancy in different countries may be due to the genetic variation of S. pneumoniae or vaccina tion coverage.In addition, the serotype distribution between adults and children was significantly different.The most predominant serotypes among adults with pneumonia were 35B, 3, 11A/D, and 23A in the United States; 3, 8, 15A, 12F, and 11A in the UK; and 12F, 3, 23A, 19A, and 10A in Japan (25)(26)(27).It is, therefore, suggested that the prevalent serotypes not covered by a vaccine should be considered in future recombinant protein vaccine research and development plans.The PCV13 serotype coverage in invasive 0.864 a Antibiotic resistance and resistance genes in 78 strains with different serotypes and CCs.The chi-square test was used to test whether the differences between two groups were statistically significant.The Bonferroni method based on the chi-square test was used to test whether the differences among multiple groups were statistically significant.According to the Bonferroni method, when P < 0.0166, the differences among three groups were statistically significant; when P < 0.0125, the differences among four groups were statistically significant."Bold" means the difference is statistically significant, and "N.A. " means no difference among multiple groups."*" indicates P < 0.05/n." # " indicates the situation where 0.05/n < P < 0.05, which means that the P value matches P < 0.05 but does not match the chi-square test with the Bonferroni method.The symbols (α, β,γ) represent the groups with significant differences.Compare multiple sets of data in pairs based on the first group in the Bonferroni method: P < 0.05/n, "γ" means significant differences; P = 0.05/n, "β" means unable to determine; P > 0.05/n, "α" means no significant differences.a, meningitis breakpoint; b, non-meningitis breakpoint.PEN, penicillin; VAN, vancomycin.Resistance genes including erm (B), mef (A), and msr (D), TET resistance gene tet (M), CHL resistance gene cat-TC, and expended-spectrum β-lactamase blaTEM-116 were antibiotic resistance-related genes.
isolates was not only higher than a multi-center study conducted in China (73.3%) but also higher than other developed countries (Spain, 41%; Japan, 28.2%) (10,27,28).Although pneumococcal-related vaccines have been introduced in China for a long time, pneumococcal vaccination is still a non-program immunization vaccine, according to the principle of informed and voluntary use.Therefore, strengthening the awareness of vaccine protection in China, especially in Jiangsu, would help reduce the S. pneumoniae infection rate.MLST analysis showed that ST271, ST320, and ST876 were the most predominant STs in our study, which were similar to the STs in other regions of China (16).The current study identified three novel STs and one novel allele, indicating that there is variation in the genetic evolution of S. pneumoniae.In the phylogenetic tree analysis, there were only two clonal complexes (CC271 and CC876) relatively less than in other regions of China (10).This phenomenon with regional features may be attributed to the individual hospital analysis rather than a multicenter study.Moreover, ST271, ST320, and ST876 were shown to be correlated with serotypes 19F, 19A, and 14, which are in agreement with the STs previously reported (16).Phylogenetic tree analysis also revealed that most ST strains manifested a good outcome; only a few ST strains, including ST320, ST271, and ST876, may lead to a poor outcome.
The status of S. pneumoniae resistance is still serious in China, which reflects the widespread use of antibiotics, the large spread of drug-resistant clones, and low vaccination coverage (7).Beta-lactam antibiotics are widely recognized as an important choice to treat invasive S. pneumoniae infection (31,32).Our study revealed the opposite result that the S. pneumoniae resistance rate to penicillin was 0% in non-meningitis patients but 100% in meningitis patients.The results suggest that penicillin could be used as a first-line antibiotic for children with invasive S. pneumoniae non-meningitis infection, rather than a meningitis infection.In agreement with previous studies, our study showed that serotype 19F strains had an apparent higher resistance rate to β-lactam antibiotics than others, which was associated with the spread of the resistant clone CC271.Apart from this, all isolates harboring erm (B) and tet (M) genes showed extremely high resistance to ERY and TET.Clearly, all isolates were sensitive to both VAN and LZD, which suggested that VAN and LZD could be alternatives for empiric treatment.
The highlight of this study was the utilization of WGS technology to determine the prevalence and molecular characteristics of invasive S. pneumoniae strains from children in Jiangsu Province, China.This study not only filled the gaps in the pediatric pneumo coccal database of Jiangsu Province but also provided a reference for clinicians in this region to treat IPD.However, this study only included one children's hospital, resulting in a small sample size with which we cannot compare annualized differences.Although the isolates from two specimen sources of each pediatric patient were confirmed to be the same serotype and ST, there were differences in the virulence gene profile and drug sensitivity results, the reasons for which need further research.
In summary, this study provided a systemic insight into the epidemiologic investiga tion of S. pneumoniae strains causing IPD in children.The rate of pneumococcal infection in our study has declined since the COVID-19 outbreak in 2019, so wearing masks could reduce the transmission of S. pneumoniae.Given the high PCV13 coverage rate and MDR full coverage, universal infant vaccination with PCV13 seems essential in this region.Finally, all isolates carried virulence genes, which could inspire recombinant protein vaccine development programs in the future.

FIG 1
FIG 1 Serotype distribution and coverage of pneumococcal vaccines among S. pneumoniae isolates.

FIG 2 7 TABLE 2 8 TABLE 2
FIG 2 Minimum-spanning tree-like structure via the goeBURST full MST algorithm.(A) goeBURST distance at SLV level 1 showing that the 27 STs were divided into 2 CCs and 19 singletons.(B) goeBURST distance at LV level 6 showing the relationship between STs and serotypes.Each disk represents an ST, and each color represents a serotype.(C) The relationship between STs and years.(D) The relationship between STs and outcomes.

FIG 3 10 TABLE 3
FIG 3Maximum likelihood tree of 78 isolates.The RAxMl tree was constructed from SNPs using the GTRGAMMA method.The genetic characteristics of all isolates were annotated.ST, orange indicates the novel STs found in this study.

TABLE 1
Clinical characteristics of 70 pediatric patients a Abandon means an outcome that pediatric patients rapidly deteriorate and parents have to give up treatment to request automatic discharge.