Streptococcus pneumoniae is a common extracellular pathogen worldwide. It often colonizes the mucosal surface of the upper respiratory tract of healthy people and is transmitted to susceptible populations through droplets, causing pneumococcal disease (PD) (1). After reaching the nasopharynx via droplets, S. pneumoniae may remain there, causing asymptomatic colonization, or may further invade the deep respiratory tract, causing pneumococcal pneumonia. Moreover, S. pneumoniae can release a variety of virulence factors, invade deep into tissue, break through the vascular endothelium, and cause pneumococcal sepsis. Furthermore, S. pneumoniae can continue to accumulate in the peripheral blood and multiply in the brain, causing pneumococcal meningitis. In clinical practice, S. pneumoniae from the nasopharynx has also been found to directly invade the brain after head trauma (2). Currently, S. pneumoniae is not only the leading cause of bacterial pneumonia in children but also the leading cause of sepsis and meningitis in children globally; this type of infection is referred to as invasive pneumococcal disease (IPD) (3).

At the end of 2019, COVID-19 was first discovered in China, and the first wave of the global pandemic occurred. From 2019 to 2022, the Chinese government implemented a series of strict population control measures to curb the spread of COVID-19, with each area adopting “zero infection” as the primary medical goal. The measures taken to cut off the transmission routes of respiratory pathogens also reduced the incidence of bacterial respiratory infections, such as pneumococcal disease, in children. However, since 2022, a second wave of the COVID-19 pandemic has occurred in various areas of mainland China despite the “early diagnosis and early isolation” strategy for COVID-19, which has lasted for more than 2 years. As population control measures been downgraded and even lifted, the incidence of transmitted respiratory bacterial infections in children, including S. pneumoniae infections, has rebounded rapidly.

Widespread vaccination with pneumococcal conjugate vaccines (PCVs) can not only reduce the infection rates of the serotypes they cover in the population but also protect against serotypes with high resistance frequencies (4). As one of the few vaccines designed to target resistant pathogens directly, the widespread use of PCVs has led to decreases in the proportions of S. pneumoniae exhibiting nonsusceptibility to penicillin, sulfamethoxazole-trimethoprim and third-generation cephalosporins on average across all regions (5). However, the increase in non-PCV13 serotypes associated with antibiotic resistance is concerning, especially during and after the COVID-19 pandemic (6).

As early as 2008, a pneumococcal polysaccharide conjugate vaccine (PCV) for children under 2 years of age was introduced in mainland China. The 7-valent pneumococcal polysaccharide conjugate vaccine (PCV-7) was subsequently delisted in mainland China in 2015, and the 13-valent pneumococcal polysaccharide conjugate vaccine (PCV-13) was launched in mainland China in 2017. However, since PCVs have not been included in the national immunization program in mainland China to date, the vaccination rates are low, as these are self-purchased vaccines that cost at least US$200 for the complete four-dose course. In 2021, the final-dose coverage of PCV-13 in mainland China was 12.47%, and in some provinces, it was only 1.17%, showing obvious interregional differences. In contrast, the final-dose coverage rates in the United States, Australia, the European Region and India were 92.0, 96.27, 82 and 69.3%, respectively (7). Therefore, Chinese children not only lack passive immunity against S. pneumoniae due to COVID-19 population control measures but also lack active immunity due to the low PCV-13 vaccination rate. During this period, changes in antibiotic susceptibility and the molecular characteristics of recirculating S. pneumoniae isolates are of concern.

In this study, we performed a cross-sectional study on PD in children during the second outbreak of COVID-19 in China. Whole-genome sequencing of S. pneumoniae isolates and antimicrobial susceptibility experiments were used to evaluate and analyze the distribution characteristics of their sensitivity to 16 antimicrobial agents, serotypes, sequence types (STs), antibiotic resistance-related genes, and virulence-related genes in Southwest China, aiming to describe the prevalence of S. pneumoniae in pediatric patients in this area.

In this study, we not only evaluated the MICs of 16 antimicrobial agents and interpreted the sensitivity breakpoints on the basis of 22 rules but also conducted whole-genome next-generation sequencing (NGS) to comprehensively analyze the molecular characteristics, including serotypes, STs, resistance-related gene carriage and variation, and virulence-related gene carriage and variation, of each pathogenic isolate in Southwest China during the second outbreak of COVID-19 that occurred in this area. Prior to this period, the government had implemented a strict “zero infection” policy for COVID-19 for several years while also eliminating the transmission routes of various other respiratory pathogens. The isolation rate of S. pneumoniae, one of the most common respiratory pathogens in children, decreased sharply during the control period. However, when this strict policy ended, the number of cases of pneumococcal infection, including invasive infections, in children began to increase again. Therefore, after 3 years of pandemic prevention and control, the development of antibiotic susceptibility and molecular characterization of pathogenic S. pneumoniae isolates has become a topic of interest and concern.

The prevalent serotypes in IPD patients were 14, 6A, and 23F, and the prevalent serotypes in NIPD patients were 19F and 6A. A significant difference in the proportion of serotype 14 strains is noted in the two groups, which was similar to the data obtained before the outbreak of COVID-19 from 2017 to 2019 (10). In the early 2020s, the prevalent serotypes of pneumococcal disease were 19F (27.2%), 6A/B (19.1%), 23F (11.0%), and 14 (8.4%) in East China (26), and the prevalent serotypes were 19F (23.4%), 23A (8.9%), 23F (7.8%), and 6B (7.8%) in Southeast China (27). Although the main prevalent serotypes of pneumococcal pathogenic isolates in China, an East Asian country with a population of 1.4 billion, were still 19A/F, 6A/B and 23A/F during this period, the proportions of subsequent specific serotypes were changing. For S. pneumoniae isolated from pediatric patients, the primary selective pressure faced by different serotypes is immunization via pneumococcal conjugate vaccines (PCVs). PCVs are self-purchased vaccines for residents in mainland China and are not included in the national medical benefits. Therefore, the PCV vaccination rate is related to local economic development and residents’ income level. Currently, the only legal PCV in mainland China is the 13-valent pneumococcal conjugate vaccine (PCV13). In Shenzhen, which is one of the four most economically developed cities in mainland China and the city closest to Hong Kong, approximately 46.6% of the S. pneumoniae isolates were not covered by serotypes of the PCV-13 vaccine (nonvaccine serotypes, NVTs) (27), and this value is higher than the values reported in other areas of mainland China. A similar “serotype replacement” phenomenon caused by vaccination is also occurring in Taiwan. Since the Taiwan authorities started vaccinating all eligible children with PCV-13 in 2015, the most prevalent serotypes of S. pneumoniae have shifted to 15A and 23A, both of which are NVTs (28). In the United States, which has a long history of PCV-13 vaccination, there has also been a significant increase in the isolation rate of NVTs 23B and 15A (29), which has also been observed in the United Kingdom, with the most prevalent serotypes being 15 B/C and 11A (30). After the COVID-19 pandemic, NVTs 24F and 8 ranked among the top three serotypes responsible for invasive infections in children in Spain (6, 31) and Portugal (32), respectively. On the one hand, the promotion and application of PCVs has indeed reduced the incidence of pneumococcal diseases in children, especially invasive pneumococcal infections resistant to first-line antibiotic treatments (5). On the other hand, long-term prevention and immunization against specific serotypes has increased the morbidity of other serotypes, forcing the development of PCVs covering more serotypes (6). PCV-20 will soon be launched in China, and PCV-24 has also entered the phase III clinical trial stage and is expected to curb the replacement of serotypes caused by PCV-13 to a certain extent. However, more than one hundred serotypes of S. pneumoniae have been discovered. The upper limit of the number of antigens and the corresponding cost of vaccine programs designed for polysaccharide capsules with different configurations are problems that must be resolved via further vaccine research and development. Perhaps in the next decade, there will be a breakthrough in research on and the development of protein vaccines or nucleic acid vaccines targeting conserved proteins or other antigenic sites of S. pneumoniae.

After the COVID-19 pandemic, CC320 (GPSC 1) remained the most prevalent CC in this area, and novel STs accounted for 11.1% of all isolates (4/36), which is higher than the number reported for children from 2017 to 2019 (6.25%) (10) but lower than the number reported for the overall population from 2018 to 2022 (35.51%) (9). These findings indicate that after 3 years of strict isolation and treatment of pneumonia patients, the genomic evolution of S. pneumoniae has not stagnated, and it still maintains a certain mutation rate. The upper respiratory tract of the healthy population is considered the main source of infection and bacterial reservoir of S. pneumoniae (1) and may also serve as the main site for the genetic recombination of S. pneumoniae. Simple population control measures that focus on controlling patients and cutting off transmission routes can reduce the infection rate of S. pneumoniae during the policy implementation period, but they are unlikely to prevent S. pneumoniae from evolving in the population and quickly returning to its original level after the isolation policy ends. Thus, measures to protect susceptible populations, mainly vaccination-based methods, appear to be more effective in preventing pneumococcal disease.

Given that the penetration ability of pneumococcal vaccines is currently insufficient, the rational use of antibiotics has become the primary measure for preventing and treating pneumococcal infection. For a long time, penicillins and cephalosporins have been the first-line antibiotics for the empirical treatment of S. pneumoniae and other transmitted bacterial respiratory pathogens in mainland China. In this study, for non-meningitis patients, regardless of whether they had invasive infections, the resistance proportions remained low, even though some pathogenic isolates were nonsusceptible to penicillin and amoxicillin. The nonsusceptibility rate to penicillins was higher than that reported in recent research in Australia (PEN: 2.6%, AMX: 2.6%) (33) and Canada (PEN: 1.4%) (34) but lower than that reported in Taiwan (PEN: 43.41%) (28) and much lower than that reported in Vietnam (PEN: 100.0%) (35).

Variation in penicillin-binding proteins (PBPs) is the leading cause of the resistance of S. pneumoniae isolates to penicillins, cephalosporins, and carbapenems. When the structures of PBPs change, these β-lactam antibiotics cannot effectively bind to them and thus cannot inhibit the formation of peptidoglycan in the cell wall, rendering bactericidal activity ineffective (36). Li et al. developed a classification system in which a pneumococcal isolate was assigned to a “PBP type” on the basis of sequence signatures in the transpeptidase domains (TPDs) of the three critical PBPs (PBP1a, PBP2b, and PBP2x) and further predicted the susceptibility of the isolates to β-lactams (24). In this study, we used the Pathogenwatch website to invoke the model for prediction, and the interpretation of penicillin (meningitis and non-meningitis) were 100% consistent with the traditional MIC identification method. However, for other β-lactam antibiotics, the prediction consistency of the PBPs method decreased, and the false positive rate for resistant strains increased. The possible reason for the decline in prediction consistency is that a considerable number of new PBPs-type strains (8/36, 22.2%) were found among the strains isolated in this study, making it difficult for the previously established prediction model to obtain accurate interpretations.

For patients with meningitis, we should consider not only the sensitivity of pathogenic isolates to antibiotics but also the ability of antibiotics to penetrate the blood–brain barrier and whether sufficient drug concentrations can be achieved in the brain. The CLSI penicillin resistance MIC breakpoint for patients with meningitis is set at 0.06 μg/ml, which is much lower than the MIC breakpoint of 8 μg/ml for patients without meningitis. In this study, according to the interpretation criteria for meningitis, almost all the isolates were penicillin resistant, and only approximately half of the isolates were susceptible to cefotaxime, ceftriaxone, and cefepime. Considering that S. pneumoniae is common in China and that the isolates are highly resistant to penicillin and third-generation cephalosporins, the Chinese guidelines recommend the use of third-generation cephalosporins plus vancomycin as the initial empirical treatment for bacterial meningitis (37).

Macrolide antibiotics, including erythromycin, were once widely abused in China given their broad antibacterial spectrum and ability to kill atypical pathogens, including Mycoplasma and Chlamydia, which has led to widespread resistance of S. pneumoniae to erythromycin. In this study, all of the isolates were resistant to erythromycin, which was similar to previous reports in China (9, 38, 39). The resistance of S. pneumoniae to macrolides occurs mainly through the targeted modification of ribosomes by the ribosomal methyltransferase encoded by erm(B), thereby reducing the binding of macrolides. The expression of erm(B) can be induced by erythromycin. The leader peptide, which causes mRNA attenuation and stabilizes its structure to prevent further translation, undergoes a change in conformation in the presence of erythromycin, allowing the expression of erm(B). In addition, mef(A/E) encodes MFS-type efflux proteins, which are located on the same operon as msr(D), which encodes an ABC-F subfamily protein. Together, they enhance the elimination of macrolides and increase resistance. In this study, almost all the isolates carried erm(B), and more than one-third of the isolates carried msr(D) and mef(A/E), indicating that both resistance mechanisms are the main causes of the high macrolide resistance of S. pneumoniae in China. Notably, after the second wave of the COVID-19 pandemic in China, from late 2023 to early 2024, Mycoplasma pneumoniae pneumonia was widely prevalent in pediatric patients, and the prevalent Mycoplasma strains were generally resistant to macrolides, forcing the use of quinolones, which exhibit joint and cartilage toxicity, for treatment (40).

The polysaccharide capsule and protein virulence factors are the two main sources of virulence in S. pneumoniae. In the virulence gene analysis, all the isolates carried capsule-related genes, and their serotypes were successfully identified. Among the other virulence genes, ply encodes pneumolysin, which is the major exotoxin of S. pneumoniae. As a cholesterol-dependent cytolysin (CDC), pneumolysin acts on cholesterol-rich biofilms, disrupts tight junctions between endothelial cells, inhibits the cilia of the bronchial epithelium, destroys the mechanical barrier of the respiratory tract, promotes the adhesion of isolates to the bronchial epithelium and accelerates infection. Moreover, pneumolysin can damage the epithelial structure of alveolar capillaries, promote isolate entry into the blood, and cause invasive pneumococcal diseases (41). In this study, ply also exhibited sequence conservation, with sequence identity among strains >99%. The pneumolysin derivative PlyD1 is safe and immunogenic and is an important candidate protein vaccine that is expected to overcome the serotype limitations of existing pneumococcal polysaccharide conjugate vaccines (42, 43).

Among the other highly conserved virulence factors, neuraminidase A, which is encoded by the NanA gene, cleaves terminal sialic acid residues from oligosaccharide receptors in host epithelial cells to facilitate colonization by S. pneumoniae (44). The lytic amidase LytA, which is encoded by the lytA gene, is a peptidoglycan hydrolase anchored on the bacterial cell wall that can hydrolyze the amide bond between N-acetylmuramic acid and L-alanine and plays a key role in bacterial autolysis (45). Hyaluronidase A, which is encoded by the hysA gene, is an LPXTG-anchored surface protein that facilitates tissue invasion by breaking down extracellular matrix (ECM) components (46). Pneumococcal surface antigen A, which is encoded by the PsaA gene, anchors to the bacterial membrane via a lipid attached to the protein and transports Mn²⁺ and Zn²⁺ into the cytoplasm (47). Pneumococcal adherence and virulence factor A, which is encoded by the pavA gene, acts directly as a fibronectin adhesin and affects pneumococcal colonization by modulating the expression or function of virulence determinants (48). The choline binding protein E, which is encoded by the pce/cbpE gene, is involved in the distribution and restructuring of the content of choline in the cell wall and indirectly modulates the activity of other CBPs and pathogen–host interactions (49). Given that these virulence factors are widely distributed and highly conserved in S. pneumoniae, they have also attracted attention in the development of vaccines and diagnostic reagents. In recent years, studies have reported that these compounds and their derivatives have strong immunogenicity and vaccine potential, can be further developed as combined antigen vaccines or fusion protein vaccines (50–53), and can be used as PCR detection targets in the diagnosis of pneumococcal disease (54, 55).

In this study, no significant difference was detected between the virulence gene carriage rates of S. pneumoniae isolates causing invasive infection and noninvasive infection, nor was there any sequence variation. However, for serotype 19F isolates, their iga carriage rate was significantly greater than that of other serotype isolates, which means that they were more likely to express IgA1 protease, which cleaves the Pro227-Thr228 peptide bond in the IgA1 hinge and may enable S. pneumoniae to subvert the antigen specificity of the humoral immune response to facilitate adhesive interactions and persistence on the mucosal surface (56). Moreover, almost all serotype 19F isolates carried both types of pili, which gives them an advantage in competing with other serotype isolates for adhesion and invasion and makes it easier for them to overcome host defenses and participate in biofilm formation (57). Therefore, this result indirectly confirms that the promotion of PCV-13 in Southwest China may be beneficial, as serotype 19F is a vaccine serotype (VT).

Our study focused on invasive infections with S. pneumoniae in pediatric patients in Southwest China from August 2022 to September 2023, when the second outbreak of COVID-19, dominated by the Omicron variant, occurred in this area. During this period, almost all medical resources were focused on the prevention, control and treatment of COVID-19. In this context, we continued to culture, purify, preserve, sequence and analyze clinically invasive and noninvasive S. pneumoniae isolates, revealing their antibiotic susceptibility and performing molecular characterization during this unique historical period. However, the primary limitation of this study is that the sample size for invasive strains was small, and the results of the analysis were mostly limited to descriptive results. Considering the timeliness of the data and despite the shortcomings of the study, the findings still provide a theoretical basis for the prevention and treatment of S. pneumoniae in this new-normal period with respect to COVID-19, and we will continue to pay attention to its epidemic status in this area.

In conclusion, this study provides valuable information on the antibiotic susceptibility and molecular prevalence of pediatric invasive and noninvasive S. pneumoniae isolates during the second outbreak of COVID-19 in Southwest China. The most prevalent serotypes in IPD patients were 14, 23F and 6A, and the prevalent CCs were CC320 (GPSC 1), CC199 (GPSC 4), and CC902 (GPSC 321). Moreover, 19F remained the most prevalent serotype in respiratory isolates. Considering the prevalence of antimicrobial resistance, a third-generation cephalosporin combined with vancomycin can be used to treat pediatric patients with invasive infections, especially meningitis. Alternatively, levofloxacin or moxifloxacin can be used instead if necessary. Benefits can be expected from vaccinating infants with pneumococcal conjugate vaccines, and conserved virulence factors may also contribute to the development of next-generation vaccines.