Complete Sequences of Six Penicillin-Binding Protein Genes from 40 Streptococcus pneumoniae Clinical Isolates Collected in Japan

Abstract

All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 g/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs м 2 g/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 g/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were м1 g/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 g/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to ␤-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with ␤-lactam resistance.

An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae : Mutational Analysis of Thr338

“…Due to this variation it is difficult to distinguish PBP mutations involved in resistance development from alterations present due to the distinct origin of the gene. Comparison of a large number of sequences from resistant clinical isolates revealed that at least one of two sites is changed in most resistant PBP2x isolates, both of which are located in the active site cavity of the enzyme: Q552E close to the K547TG triad and T338A(P,G) within the S337TMK motif (3,7,14,43,44,47,54,57). Curiously, most of the many PBP2x mutations identified in spontaneous cefotaxime-resistant laboratory mutants are distinct from changes seen in clinical isolates, including the T338 mutation (32), suggesting that other selective forces are acting outside the laboratory.…”

Rapid Identification of Penicillin and Macrolide Resistance Genes and Simultaneous Quantification of Streptococcus pneumoniae in Purulent Sputum Samples by Use of a Novel Real-Time Multiplex PCR Assay

“…The resistance pbp1a primers were designed to target four amino acid substitutions (Thr5743Asn, Ser-5753Thr, Gln-5763Gly, and Phe-5773Thr) that are common to all penicillin G (PCG)-intermediate and -resistant isolates (29). The resistance pbp2x primers were designed to target amino acid substitutions in the 337STMK motif, and the resistance pbp2b primers were designed to target amino acid substitutions close to the 448SSN motif (25,28). The primers for amplification of the ermB (224-bp) and mefA (294-bp) genes were gleaned from a published sequence (21).…”

Emergence of a Unique Penicillin-Resistant Streptococcus pneumoniae Serogroup 35 Strain

“…The RESIST group showed 96% sequence identity with two strains registered in PubMed, SP00080 and SP00081 (31). When amino acid sequences were compared, the RESIST group strains showed 99% identity to SP00080, 98% identity to SP00081, and 99% identity to unregistered strains J1 and J88 (4).…”

“…2) (13). The RESIST group strains showed 100% sequence identity in this resistant region to the PRSP SP00091, S. oralis 5296, and Streptococcus mitis B6 strains and 99% sequence identity to Streptococcus sanguis 1907 and PRSP DN87/577 (Table 2) (11,13,31). The pbp2b gene in the RESIST group strains contained a second resistant block that was not associated with PRSP probe 2.…”