ASSOCIATION OF A THR-371 SUBSTITUTION IN A CONSERVED AMINO-ACID MOTIFOF PENICILLIN-BINDING PROTEIN 1A WITH PENICILLIN RESISTANCE OF STREPTOCOCCUS-PNEUMONIAE

We determined the nucleotide sequence between 1,903 and 3,097 bp of pb p1a, which encodes the transpeptidase domain of PBP 1A, from clinical isolates of penicillin-resistant Streptococcus pneumoniae (PRSP) serot ypes 19 (n = 8), 6 (n = 9), 23 (n = 6), and 14 (n = 2) and two penicil lin-susceptible S. pneumoniae (PSSP) isolates. These serotyped PRSP st rains were isolated predominantly in Japan from 1993 through 1997. The 25 resistant strains were classified into five groups on the basis of the extent of sequence differences. Strains in groups I (n = 5; serot ype 6), II (n = 3; serotype 19), and III (n = 12; different serotypes) had sequences highly homologous to the sequence of pbp1a of PRSP stra ins from South AG-ica, Spain, and the United States. The group TV stra in (n = 1; serotype 14) had unique deletions from or insertions in the sequences. The sequences of group V strains (n = 4; serotypes 6 and 2 3) had relatively few differences from the sequences of the PSSP strai ns. For strains (n = 18) for which the threonine at codon 371 (Thr-371 ) in a conserved STMK motif of PBP 1A was substituted with an alanine or a serine (in addition to having altered pbp2x and pbp2b genes), pen icillin MICs were greater than or equal to 1.0 mu g/ml. The PBPs 1A of these strains showed decreased affinities for [H-3]benzylpenicillin a nd slightly faster mobilities on sodium dodecyl sulfate-polyacrylamide gels. In contrast, for strains (n = 4) without a substitution at Thr- 371 in PBP 1A but with mutations in both pbp2x and pbp2b, penicillin M ICs were 0.125 to 0.25 mu g/ml, and the affinities of their PBPs 1A we re similar to that of PSSP PBPs LA. Furthermore, for the Thr-371-subst ituted strains (n = 3) with altered pbp2x genes but native pbp2b genes , penicillin MICs were 0.125 to 0.25 mu g/ml. These results suggest th at amino acid substitution of Thr-371 contributes to the development o f penicillin resistance in PRSP strains with altered pbp2x and pbp2b g enes.