First Molecular Characterization of Group B Streptococci with Reduced Penicillin Susceptibility

Strains and identification of GBS.The properties of 3 control strains and 14 strains of GBS with reduced penicillin susceptibility (PRGBS) isolated clinically are listed in Table 1. Nine PRGBS strains were isolated from 1995 to 1998 and were kept until this study. Streptococcus pneumoniae ATCC 49619 was a quality control strain used for measurement of the MICs of the antimicrobial agents tested. To obtain candidate PRGBS strains, we screened all 159 clinical isolates sent to a clinical laboratory from various Japanese clinical facilities over 3 days in 2005. We initially predicted that the PRGBS candidates would show reduced susceptibility to some β-lactams as a result of mutations in penicillin-binding protein (PBP) genes, since penicillin-resistant strains of Streptococcus pneumoniae usually demonstrate resistance to oxacillin. Thus, we selected from among the 159 clinical isolates 49 isolates which showed oxacillin MICs of >2 μg/ml by the microdilution method. We further selected from among the 49 isolates 5 isolates that demonstrated high levels of resistance to β-lactams. We analyzed both the nine clinical isolates stocked before 1998 and the five new clinical strains isolated in 2005. All 14 strains were isolated from the sputa of individual patients, most of whom were elderly patients, at 12 geographically separate hospitals in Japan from 1995 to 1998 and in 2005. None of the PRGBS candidates were isolated from sterile body sites such as blood or cerebrospinal fluid. All clinical isolates were subjected to multiple tests for the exact identification of GBS, including Gram staining, 16S rRNA PCR followed by restriction enzyme-digested fingerprinting, biochemical identification, surface antigen detection and the CAMP test, with Streptococcus agalactiae ATCC BAA-611 and S. agalactiae ATCC 12403 used as the positive controls. PCR amplification of 16S rRNA and restriction enzyme-digested fingerprinting were performed as described previously (18). Biochemical identification was performed with the API 20 Strep system (bioMérieux) and surface antigen detection was performed by the Slidex strep test (bioMérieux) according to the manufacturer's instructions. Serotyping was performed with anti-GBS serotype-specific serum (Denka Seiken). Notably, 8 of the 14 strains were serotype III (Table 1), a well-known major serotype found in strains causing neonatal meningitis in Western countries, although serotypes VI and VIII are the predominant types among Japanese clinical isolates.

Measurement of MICs.Measurement of the MICs of penicillin G, penicillin V, ampicillin, oxacillin, cefazolin, cefepime, cefotaxime, ceftizoxime, and meropenem were performed by the agar dilution method, as recommended by the CLSI (6). A penicillin-susceptible S. pneumoniae strain, ATCC 49619, was used as the quality control strain for measurement of the exact MICs.

PFGE.Pulsed-field gel electrophoresis (PFGE) of the 14 clinical isolates of PRGBS was performed as described previously (13), with minor modifications. The restriction enzyme used in the PFGE experiment was ApaI. Run conditions were generated by the autoalgorithm mode of the CHEF Mapper PFGE system with a size range of 20 to 200 kb.

Sequencing of high-molecular-weight PBP genes.Five genes encoding high-molecular-weight PBPs (PBP 1A, PBP 1B, PBP 2A, PBP 2B, and PBP 2X) were amplified by DNA polymerase Pyrobest (Takara) by using genomic DNA as the template. The PCR conditions were as follows: 1 cycle of 98°C for 1 min; 30 cycles of 98°C for 10 s, 55°C for 1 min, and 72°C for 2 min 30 s; and 1 cycle of 72°C for 7 min. The mixture was then held at 4°C. Purified PCR products were obtained with a Wizard SV gel and PCR clean-up system (Promega), followed by sequencing reactions and analysis with an ABI Prism 3100 genetic analyzer (Applied Biosystems). The primers used for amplification of the PBP genes and sequencing are listed in Table 2.

Visualization and Western blotting analysis of PBPs.Membrane fractions were prepared from the ATCC strains and the clinical isolates as described previously (24). The membrane fraction (400 μg) was incubated with 12.5 mM of fluorescence-conjugated penicillin V (Bocillin FL; Molecular Probes) at 37°C for 30 min and a 3× sodium dodecyl sulfate (SDS) sample buffer was added, followed by SDS-polyacrylamide gel electrophoresis (PAGE) analysis with a fluorescent image analyzer (LAS-3000 multicolor; Fuji).

Western blotting analysis was performed with rabbit anti-PBP 2X serum raised against a keyhole limpet hemocyanin-conjugated peptide corresponding to the 14 amino acid residues of PBP 2X (amino acids 237 to 250), which is a very conserved region that lacks mutations even in PRGBS.

Allelic exchange experiments.To generate S. agalactiae ATCC BAA-611 integrant strains harboring the chromosomally encoded PBP 2X genes derived from the clinically isolated PRGBS strains, we performed allelic exchange experiments, as described previously (4). A thermosensitive Escherichia coli-streptococcus shuttle vector, pG+host6, was modified by removing the Eam1105I-Aat II fragment to delete its penicillinase gene, and the resultant vector was designated pG+host6Δamp. To generate targeting vectors, fragments containing the nucleotide region from positions 295821 to 297819 of the GBS ATCC BAA-611 genome were amplified from the chromosomal DNA of clinical isolates B12 and B503 with Pyrobest DNA polymerase (Takara) and ligated into pG+host6Δamp. Several inserts were sequenced, and no additional mutation was found in the inserts. Electrocompetent cells were made from S. agalactiae ATCC BAA-611 as described previously (12). One microgram of the targeting vector was introduced into electrocompetent cells derived from GBS strain BAA-611, as described previously (12), and transformants were selected on agar plates containing 0.5 μg/ml of erythromycin at 30°C. To obtain integrant cells in which the targeting vectors had integrated into the chromosomes of the recipient GBS cells, transformants were incubated in liquid medium containing 5 μg/ml of erythromycin at 37°C, as described elsewhere (4). Such integrant strains were successively cultivated for 3 days in liquid medium at 30°C without erythromycin selection to facilitate the excision of vector pG+host6Δamp. Integrant strains were selected on an agar plate containing 4 μg/ml of ceftizoxime, and susceptibility to erythromycin was confirmed by use of an agar plate containing 5 μg/ml of erythromycin. The presence of the altered PBP 2X gene in each integrant strain was confirmed by sequencing, and the integrant strains were subjected to MIC measurements.

Nucleotide sequence accession numbers.All high-molecular-weight PBP genes derived from the 14 clinical isolates were deposited in the EMBL/GenBank database through the DDBJ database and were assigned accession numbers AB279794 to AB279863.

Confirmatory identification of GBS.From among the 159 clinical strains isolated in 2005, we selected 49 isolates for which the oxacillin MICs were >2 μg/ml by the microdilution method. We also selected from among these 49 isolates 5 isolates that demonstrated high-level resistance to β-lactams. We analyzed nine stocked from 1995 to 1998 and five clinical isolates isolated in 2005. The identities of the 14 clinical isolates were confirmed to be S. agalactiae by Gram staining, 16S rRNA PCR followed by restriction enzyme-digested fingerprinting (18), biochemical identification, surface antigen detection, and the CAMP test.

These 14 strains were gram-positive cocci, and no apparent morphological abnormalities were observed at the light microscopy level when the strains were cultured without penicillins (data not shown). The partial 16S RNA gene amplified from chromosomal DNA was fingerprinted with the HaeIII restriction enzyme. The fingerprinting patterns of the 14 strains were identical to those of the standard strains of S. agalactiae obtained from ATCC (ATCC BAA-611 and ATCC 12403) (data not shown). All 14 clinical isolates showed biochemical properties consistent with those of S. agalactiae. We further performed the streptococcus grouping latex agglutination test with anti-streptococcus-specific surface antigen serum and confirmed the validity of the identification. Finally, the CAMP test, a GBS-specific identification test which detects the production of GBS-specific CAMP factor, confirmed that all the clinical isolates showed the typical phenotype of GBS. The several assays mentioned above clearly showed that these strains must be S. agalactiae.

PFGE.We performed PFGE with the 14 clinical isolates (Fig. 1). All clinical isolates except B10 and B12 showed different DNA band patterns, suggesting that all clinical isolates except B10 and B12 were genetically nonrelated strains. Interestingly, B10 and B12, which were clinical isolates from the same hospital, showed very similar DNA band patterns, implying their clonal relatedness.

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FIG. 1.

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

Measurement of β-lactam MICs.We determined the MICs of several β-lactams for the 14 strains by the agar dilution method recommended by the CLSI (Table 3). The MICs for the control strain, S. pneumoniae ATCC 49619, fulfilled the criteria for “quality control” established by the CLSI. All 14 strains, however, showed reduced susceptibilities to penicillin G (0.25 to 1 μg/ml), oxacillin (2 to 8 μg/ml), and ceftizoxime (4 to 128 μg/ml). Some strains were also “insusceptible” to ampicillin (0.12 to 0.5 μg/ml), cefazolin (0.5 to 2 μg/ml), cefepime (0.25 to 1 μg/ml), and cefotaxime (0.12 to 2 μg/ml).

Genes for high-molecular-weight PBPs.Alterations in the high-molecular-weight PBPs are widely acknowledged to be the leading molecular mechanisms of resistance to β-lactams in S. pneumoniae. To obtain deduced amino acid sequences of the PBPs of the 14 GBS strains, we analyzed the nucleotide sequences of the coding regions for five high-molecular-weight PBPs (PBP 1A, PBP 1B, PBP 2A, PBP 2B, and PBP 2X) (Fig. 2). Some of the 14 clinical isolates possessed a few mutations in PBP 1A (0 to 3 amino acid substitutions and 0 to 4 amino acid deletions and 0 to 10 nucleotide substitutions and 0 to 12 nucleotide deletions), PBP 1B (0 to 1 amino acid substitution and 0 to 4 nucleotide substitutions), PBP 2A (0 to 2 amino acid substitutions and 2 to 11 nucleotide substitutions), and PBP 2B (0 to 1 amino acid substitution and 0 to 2 nucleotide substitutions) (Fig. 2A). On the other hand, all 14 clinical isolates possessed several deduced amino acid substitutions in PBP 2X (2 to 7 amino acid substitutions and 2 to 10 nucleotide substitutions) adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively), considered to form the active site of the enzyme (Fig. 2B and C). PBP 2X of PRGBS did not have a mosaic structure like that of the PBPs of penicillin-resistant S. pneumoniae strains. The amino acid substitutions found in PBP 2X of PRGBS could be classified into two types, Q557E and V405A. Among the 14 clinical isolates, all isolates except isolate B7 possessed at least one of these two amino acid substitutions, which existed adjacent to the conserved active-site motifs of PBP 2X. The Q557E of PBP 2X in PRGBS corresponded to the Q552E of PBP 2X found in penicillin-resistant S. pneumoniae strains because the amino acid sequence of PBP 2X in GBS showed 74% similarity to that in S. pneumoniae R6, and the three active-site motifs in PBP 2X were well conserved between the GBS strains and S. pneumoniae. These results indicate that alterations in PBP 2X existing around the regions corresponding to the active-site motifs are the critical determinants responsible for the reduced susceptibility to penicillin G, oxacillin, and ceftizoxime among the penicillin-insusceptible GBS strains.

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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.

Visualization and identification of PBPs.To elucidate the resistance mechanisms on a molecular basis, we next visualized the PBPs using fluorescent-conjugated penicillin V, Bocillin FL. Membrane fractions prepared from the 14 clinical strains and the 2 ATCC strains were incubated with Bocillin FL, followed by SDS-PAGE and detection with a fluorescent image analyzer (Fig. 3A and B). The two ATCC strains possessed six apparent PBPs, but one band was eliminated from among all 14 clinical strains, demonstrating penicillin insusceptibility. To identify the eliminated band, we generated rabbit anti-PBP 2X serum by immunization with a synthetic peptide corresponding to the 14 amino acid residues of PBP 2X (amino acids 237 to 250), which is a very conserved region lacking mutations even in PRGBS. Western blotting analysis of the membrane fraction prepared from the two ATCC strains revealed that the band that was commonly eliminated among the 14 clinical strains corresponded exactly to PBP 2X (data not shown).

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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.

Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum revealed that the level of production of PBP 2X among the 14 clinical isolates was reduced or eliminated, although the two ATCC strains possessed a significant amount of PBP 2X (Fig. 3C).

Allelic exchange with PRGBS-derived PBP 2X gene.Finally, to assess the contribution of the altered PBP 2X genes to the elevation of penicillin insusceptibility, the PBP 2X genes derived from penicillin-insusceptible clinical isolates were introduced into a β-lactam-susceptible GBS strain, strain ATCC BAA-611, through allelic exchange experiments. One clinical isolate with either type of altered PBP 2X, strain B12 for the Q557E type and strain B503 for the V405A type, was selected, because these strains showed the least number of alterations in the PBP 2X genes. Each type of altered PBP 2X gene from a penicillin-insusceptible strain was transformed into ATCC BAA-611 by integration (Table 4). The MICs for these integrated strains revealed that the reduced susceptibilities to penicillin G, oxacillin, and ceftizoxime were comparable to those of the parental clinical isolates. These results confirmed that both types of altered PBP 2X genes (types Q557E and V405A) are the major determinants of reduced susceptibility to penicillin G, oxacillin, and ceftizoxime in GBS.