First molecular characterization of group B streptococci with reduced penicillin susceptibility

Abstract

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 microg/ml, 2 to 8 microg/ml, and 4 to 128 microg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of beta-lactam-insusceptible GBS from a phenotypic standpoint.

FIG. 1.

PFGE of 14 clinical isolates (isolates in lanes B1 to B516). Lanes M, molecular markers.

FIG. 2.

Deduced amino acid sequences of high-molecular-weight PBPs of control strains (strains ATCC BAA-611 and ATCC 12403) and clinical isolates (isolates B1 to B516). (A and B) Deduced amino acid sequences of high-molecular-weight PBPs (PBP 1A, PBP 1B, PBP 2A, PBP 2B, and PBP 2X). The numbers above the sequences indicate the position number of the amino acid sequence. The blanks and dashes indicate no substitution and deletional change of amino acid, respectively. (C) Pattern diagram of PBP 2X. Common amino acid substitution types V405A and Q557E exist adjacent to active site motifs SSN and KSG, respectively.

FIG. 3.

Visualization of PBPs and Western blotting of PBP 2X from control strains (strains ATCC BAA-611 and ATCC 12403) and clinical isolates (isolates B1 to B516). (A and B) Visualized PBPs. Membrane fractions derived from strains were incubated with fluorescent-conjugated penicillin, followed by SDS-PAGE on a 10% (A) or 6% (B) polyacrylamide gel. Arrows indicate the band corresponding to PBP 2X. (C) Western blotting of PBP 2X. Western blotting analysis was performed with membrane fractions derived from strains and rabbit anti-PBP 2X serum.