Serotype IX, a Proposed New Streptococcus agalactiae Serotype

Abstract

We identified three isolates of Streptococcus agalactiae (group B streptococcus [GBS]), of human origin, which failed to react with antisera against any of the nine known GBS serotypes. Polyclonal rabbit antisera raised against these isolates and standard GBS typing sera were used in capillary precipitation and Ouchterlony tests to compare the strains with known GBS serotype reference strains. All three previously nontypeable isolates reacted with all three new antisera, producing lines of identity in the Ouchterlony test. Weak cross-reactions with antisera against several GBS serotypes were observed but were removed by absorption with corresponding antigens. The new antisera were used to test 227 GBS isolates that had been nontypeable or difficult to type using standard antisera. Of these, five reacted with the new antisera. These results suggested that all eight isolates belong to the previously unrecognized GBS serotype. They were tested by Western blotting for the Calpha and Cbeta proteins and by PCR to identify molecular serotypes and surface protein antigen genes. Two segments of the cps gene cluster (3' end of cpsE-cpsF and 5' end of cpsG, approximately 700 bp; 3' end of cpsH and 5' end of cpsM, approximately 560 bp) were sequenced. All eight isolates expressed Calpha, and seven expressing the Cbeta protein and the corresponding genes, bca and bac, respectively, were identified. They all share the same, unique partial cps sequence. These results indicate that these eight isolates represent a new S. agalactiae serotype, which we propose should be designated serotype IX.

FIG. 1.

Three Ouchterlony tests (A, B, and C) are shown. Ten microliters antiserum was added in the center hole, and 10 μl of either 0.1 N or 0.2 N extract antigen was added in the surrounding holes. (A) Antiserum raised against isolate 22634 was added in the center hole. Reactions appeared toward extract antigens from all three isolates. A second line appeared towards extract antigen of isolate 22634, and this reaction also appears with GBS type Ib where there is line of identity. (B) Antiserum (raised against 22634) absorbed with antigen from GBS type Ib was added in the center hole. Only reactions against antigen of the three isolates appeared. (C) Antiserum raised against GBS type Ib was added in the center hole. A double line appeared against extract antigen (0.1 N) of GBS type Ib.

FIG. 2.

Differences between proposed serotype IX cpsH-cpsM gene and amino acid sequences and those of serotype V. (A) Gene sequence comparison between proposed serotype IX, cpsH/cpsM, accession no. EF157290 (upper lines) and serotype V, cpsH, accession no AF349539 (lower lines). Five differing sites were found (bold and underlined). The numbers on each row represent the base positions on relevant GenBank sequences. (B) Amino acid comparison between proposed serotype IX, CpsH, accession no. EF157290 (upper lines) and serotype V, CpsH, accession no. AF349539 (lower lines). Two differing sites were found (bold and underlined). The numbers on each row represent the position on relevant GenBank CpsH amino acid sequences.