Molecular characterization of nontypeable group B streptococcus

Abstract

Traditionally, the capsular polysaccharide (CPS) antigen has been used to distinguish between the nine known serotypes of group B streptococcus (GBS) by classical antibody-antigen reactions. In this study, we used PCR for all CPSs and selected protein antigens, multilocus sequencing typing (MLST), and pulsed-field gel electrophoresis (PFGE) to molecularly characterize 92 clinical isolates identified as nontypeable (NT) by CPS-specific antibody-antigen reactivity. The PCR and MLST were performed on blinded, randomly numbered isolates. All isolates contained the cfb gene coding for CAMP factor. While most (56.5%) contained a single CPS-specific gene, 40 isolates contained either two or three CPS-specific genes. Type V CPS-specific gene was present in 66% of the isolates, and all serotypes except types IV, VII, and VIII were represented. Most (44.5%) of the isolates contained a single protein antigen gene (bca, bac, rib, alp1, or alp3), and the remaining isolates had multiple protein antigen genes. Of the 61 isolates that had the V CPS-specific gene, 48 (78.6%) had the alp3 gene. PFGE analysis classified the isolates into 21 profile groups, while MLST analysis divided the isolates into 16 sequence types. Forty-two (69%) of 61 isolates with the V CPS-specific gene were in PFGE profile group 4; 41 of these 42 were sequence type 1 by MLST. These data shed new light on the antigenic complexity of NT GBS isolates, information that can be valuable in the formulation of an effective GBS vaccine.

FIG. 1.

UPGMA dendrogram showing the genetic relationship between the 16 STs. The allelic profile of each ST is shown in parentheses next to the ST designation. The number of isolates found in each ST is shown in brackets. BURST analysis identified four clonal group complexes: group 1 had STs 8, 10, 12, and 239; group 2 had STs 19 and 28; group 3 had STs 1, 44, and 238; and group 4 had STs 17, 31, and 237. The ancestral genotype of the three groups (12, 1, and 17) is denoted by an asterisk. The numbers at the nodes represent the percentage of times each node was represented in the bootstrap analysis.

FIG. 2.

PCR assay on selected nontypeable group B streptococcus performed with type Ia capsular polysaccharide-specific primer (top panel) and type V capsular polysaccharide-specific primer (bottom panel). Isolates 32 and 34 to 37 were scored positive for possessing genes for both type Ia and type V capsular polysaccharides. Ia and V denote the control DNA from Ia strain 090 and type V strain CJB111. The PCR product sizes were 354 bp and 374 bp for type Ia and type V capsular polysaccharide-specific genes, respectively.