Genotyping of the capsule gene cluster (cps) in nontypeable group B streptococci reveals two major cps allelic variants of serotypes III and VII

Abstract

Forty group B Streptococcus (GBS) isolates obtained from Europe and the United States previously reported to be nontypeable (NT) by capsule serotype determination were subjected to buoyant density gradient centrifugation. From nearly half of the isolates capsule-expressing variants could be selected. For characterization of the remaining NT-GBS isolates, the capsule operon (cps) was amplified by the long-fragment PCR technique and compared by restriction fragment length polymorphism (RFLP) analysis. The patterns from serotype reference isolates (n = 32) were first determined and used as a comparison matrix for the NT-GBS isolates. Using two restriction enzymes, SduI and AvaII, cluster analysis revealed a high degree of similarity within serotypes but less than 88% similarity between serotypes. However, serotypes III and VII were each split in two distant RFLP clusters, which were designated III(1) and III(2) and VII(1) and VII(2), respectively. Among the isolates that remained NT after repeated Percoll gradient selections, two insertional mutants were revealed. Both were found in blood isolates and harbored insertion sequence (IS) elements within cpsD: one harbored IS1548, and the other harbored IS861. All other NT-GBS isolates could, by cluster analysis, be referred to different serotypes by comparison to the RFLP reference matrix. In pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA, patterns from allelic type 1 and 2 isolates were essentially distributed in separate clusters in serotypes III and VII. A covariation with insertion sequence IS1548 in the hylB gene was suggested for serotype III, since allelic type III(1) harboring IS1548 in hylB, clustered separately. The variation in serotype VII was not dependent on the presence of IS1548, which was not detected at any position in the type VII chromosome.

FIG. 1

Open reading frames in the GBS capsule gene cluster and positions for long-fragment PCR primers. Positions are based on two entries in the GenBank: for serotype Ia, accession no. AB028896 (39), and for serotype III, accession no. AF163833. Restriction sites for the enzymes used in the RFLP, investigation, AvaII and SduI, are denoted above the genes.

FIG. 2

RFLP analyses of long-fragment PCR amplicons from the cps capsule gene cluster from two reference type strains each for every GBS serotype. Allelic variants for serotypes III and VII are designated 1 and 2. M, molecular weight standard in kilobases.

FIG. 3

Gel-dendrogram diagram of cps long-fragment PCR amplicon RFLP from 19 NT-GBS and 32 typeable GBS isolates. Each isolate is represented by one lane where the schematic band profile is combined from separate digestions with AvaII (left) and SduI (right). The horizontal bar (upper left) represents the percent similarity coefficient. Clustering was performed using the UPGMA method.

FIG. 4

Analysis of SmaI-digested chromosomal DNA from GBS serotype III s.l. NT/PV III denotes NT-GBS where phase variant serotype III was found after gradient centrifugation; NT/CT III, nonreverting isolates with cluster type III. III₁ and III₂, allelic types III₁ and III₂, respectively, according to cps typing. (A) PFGE gel. M, molecular weight standards in kilobases. (B) Cluster analysis and presence of IS1548 in the hylB gene. The horizontal bar (upper left) represents the percent similarity coefficient. Clustering was performed using UPGMA method.

FIG. 5

Analysis of SmaI-digested chromosomal DNA from GBS serotype VII reference strains. VII₁ and VII₂, allelic variants. (A) PFGE gel. M, molecular weight standards in kilobases. (B) Cluster analysis and presence of IS1548 in the hylB gene.

FIG. 5

Analysis of SmaI-digested chromosomal DNA from GBS serotype VII reference strains. VII₁ and VII₂, allelic variants. (A) PFGE gel. M, molecular weight standards in kilobases. (B) Cluster analysis and presence of IS1548 in the hylB gene.

FIG. 6

Schematic diagram of IS elements in the capsule gene cluster in two NT-GBS blood isolates. (A) Isolate GA4096 harboring IS861 in cpsD. (B) Isolate Ros106 harboring IS1548 in cpsD. The sequences for the direct repeats (DR) at target sites are shown in boxes. The inverted repeat (IR) sequences for the present isolates are noted in open boxes and were identical with sequences previously reported for IS861 (accession no. M22449) (30) and IS1548 (accession no. Y14270) (8). Arrows show directions of transcription for IS elements.

FIG. 6

Schematic diagram of IS elements in the capsule gene cluster in two NT-GBS blood isolates. (A) Isolate GA4096 harboring IS861 in cpsD. (B) Isolate Ros106 harboring IS1548 in cpsD. The sequences for the direct repeats (DR) at target sites are shown in boxes. The inverted repeat (IR) sequences for the present isolates are noted in open boxes and were identical with sequences previously reported for IS861 (accession no. M22449) (30) and IS1548 (accession no. Y14270) (8). Arrows show directions of transcription for IS elements.