Streptococcus pneumoniae is the major causative agent of community-acquired pneumonia [[1], [2], [3]], acute otitis media [4], and invasive pneumococcal diseases (IPDs) [5,6]. Although the pathogen produces various substances related to pathogenicity, the polysaccharide capsule is the most important factor for the invasion of the pathogen into sterile sites in humans [7,8]. Currently, 100 capsule types have been reported as serotypes [9,10]. Based on these polysaccharide antigens, two vaccines, 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPSV23), have been developed and applied worldwide as a tool to prevent pneumococcal infections.

In Japan, PVC13 was incorporated into the vaccine schedule for children under the age of 5 in 2014. PPSV23 has been routinely administered to individuals over 65 since 2014. PCV13 vaccination for individuals between the ages of 6 and 65 with at risk was approved in 2021, in addition to PPSV23 approved previously. PCV13 vaccination for children sharply decreased the incidence of IPDs [11], pneumonia [12], and otitis media [13]. However, in children with immunological and physical risks, despite PCV13 vaccination, pneumococcal infections caused by non-vaccine type (NVT), not included in the vaccine type (VT) of PCV13, have gradually increased [14]. NVT has also increased significantly in pneumococcal infections in individuals age 65 and over, who have been vaccinated with PPSV23 [15].

The resistance mechanism of penicillin (PEN)-non-susceptible S. pneumoniae is related to the decrease in the affinity of β-lactam antibiotics for PEN-binding proteins (PBPs) included in peptidoglycan synthesis. Of the six PBPs identified in S. pneumoniae, abnormal PBP1A, PBP2X, and PBP2B encoded by the respective pbp genes are known to be essential for the development of β-lactam resistance [16,17].

PBP1A is an enzyme that elongates the peptidoglycan cell wall in the long axis direction; PBP2X is involved in septal synthesis; and PBP2B is involved in the formation of the lancet shape, which is typical for this bacterium. PEN and carbapenem antibiotics have high affinity for PBP1A and PBP2B, whereas cephalosporin agents have high affinity for PBP2X [18]. The resistance level, which is the decrease in susceptibility to β-lactam agents, depends on the combination of abnormal PBPs. The resistance level is low when only one gene is associated with abnormal PBPs, but when the abnormal genes overlap, the resistance level is elevated owing to the synergistic effect. High-molecular-weight PBPs have both transglucosylase (TG) and transpeptidase (TP) activities [19].

The emergence of PEN-non-susceptible S. pneumoniae with abnormal PBPs due to pbp gene mutations in the NVT isolates is expected to create therapeutic problems in the future. However, few studies have been conducted on β-lactam resistance mechanisms. In this study, we performed nucleotide sequence analysis of the pbp1a, pbp2x, and pbp2b genes involved in the resistance of NVT strains isolated from IPDs and investigated their amino acid (AA) substitutions. We i) calculated the AA substitution rates in PBPs and their pbp gene mutation rates, ii) compared the mutation rates in the strains of the same serotype, iii) identified the clonal complexes and sequence types, and iv) conformed the positioning of AA substitutions in each PBP via three-dimensional (3D) structure homology modeling.