Genetic heterogeneity in pbp genes among clinically isolated group B Streptococci with reduced penicillin susceptibility

Abstract

The recent emergence of group B streptococcal isolates exhibiting increased penicillin MICs at the Funabashi Municipal Medical Center and other hospitals in Japan prompted a comparative analysis of the penicillin-binding proteins (PBPs) from those strains with the PBPs from penicillin-susceptible strains comprising four neonatal invasive strains isolated from 1976 to 1988 and two recent isolates. The PBP sequences of the penicillin-susceptible strains were highly conserved, irrespective of their isolation date. Of six strains with reduced susceptibility to penicillin (penicillin MICs, 0.25 to 0.5 mug/ml), strains R1, R2, R5, and R6 shared a unique set of five amino acid substitutions, including V405A adjacent to the (402)SSN(404) motif in PBP 2X and one in PBP 2B. The remaining two strains, R3 and R4, shared several substitutions, including Q557E adjacent to the (552)KSG(554) motif in PBP 2X, in addition to the substitutions in PBP 2B, which are commonly found among penicillin-insusceptible strains. Strains R7 and R8, which had a penicillin MIC of 1 mug/ml, shared a unique set of eight amino acid substitutions (two in PBP 2X; two in PBP 2B, including G613R adjacent to the (614)KTG(616) motif; three in PBP 1A; and one in PBP 2A), and the Q557E substitution in PBP 2X was common to R3 and R4. The binding of Bocillin FL was reduced or not detected in some PBPs, including PBP 2X of penicillin-insusceptible strains, but no significant reduction in the level of pbp2x transcription was found in such strains. The results of phylogenetic comparative analyses imply the absence of epidemic penicillin-insusceptible strains, and several genetic lineages of penicillin-insusceptible strains have been independently emerging through the accumulation of mutations in their pbp genes, especially in pbp2x.

FIG. 1.

Nucleotide phylogram of pbp genes from GBS strains and S. agalactiae strains 2603 V/R, NEM316, and COH1. Penicillin-insusceptible strains R1 to R8 are shaded. The nucleotide sequences were aligned by using the Clustal W program and were subjected to phylogenetic analysis by the neighbor-joining method based on Kimura's two-parameter model distance matrices with the MEGA program (version 3.1). The resulting trees were bootstrapped 500 times, and the bootstrap values are shown as percentages. The scale bars indicate the expected number of changes per sequence position.

FIG. 2.

Deduced amino acid substitutions in five high-molecular-mass PBPs from GBS isolates. Residues mutated in PBPs from penicillin-insusceptible strains are shaded. Strain 2603 V/R is ATCC BAA-611, and strain NEM316 is ATCC 12403. PEN, penicillin; AMP, ampicillin; CTX, cefotaxime.

FIG. 3.

Binding of penicillin to GBS PBPs and immunodetection of PBP 2X. Membrane proteins were incubated with Bocillin FL, separated on a 10% (A) or a 5% (B) SDS-polyacrylamide gel, and detected by fluorography, as described in Materials and Methods. The molecular sizes of the protein standards (Precision Plus; Bio-Rad Laboratories, Hercules, CA) are provided on the left. (C) Membrane proteins separated on a 5% SDS-polyacrylamide gel were subjected to Western blotting with anti-PBP 2X antibody as the primary antibody, as described in Materials and Methods. Arrows indicate the position of PBP 2X. VR, S. agalactiae 2603 V/R.

FIG. 4.

PFGE profiles of genomic DNA of penicillin-insusceptible (A) and -susceptible (B) GBS isolates digested with SmaI. Lanes M, bacteriophage lambda DNA ladder as molecular size markers; VR, S. agalactiae 2603 V/R; NE, S. agalactiae NEM316.