Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae

In Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. Pneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to β-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. In PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. NVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.