Serotype identification of group B streptococci by PCR and sequencing

Abstract

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (cps) gene clusters to further define partial cps gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.

FIG. 1.

Molecular serotype identification based on the sequence heterogeneity of the 790-bp fragment (positions 1437 to 2226, corresponding to the regions of the amplicon obtained with primer pair cpsES3-cpsGA1) at the 3′ end of cpsE-cpsF and the 5′ end of cpsG (the relevant primers are indicated). Numbering start point 1 refers to start point 1 of the sequence with GenBank accession number AF332908 (for serotype reference strain 3139). Dashes indicate the same sequence as the consensus sequence; *, for serotype Ib, position 2221 refers to the sequence with GenBank accession number AB050723.

FIG. 1.

Molecular serotype identification based on the sequence heterogeneity of the 790-bp fragment (positions 1437 to 2226, corresponding to the regions of the amplicon obtained with primer pair cpsES3-cpsGA1) at the 3′ end of cpsE-cpsF and the 5′ end of cpsG (the relevant primers are indicated). Numbering start point 1 refers to start point 1 of the sequence with GenBank accession number AF332908 (for serotype reference strain 3139). Dashes indicate the same sequence as the consensus sequence; *, for serotype Ib, position 2221 refers to the sequence with GenBank accession number AB050723.

FIG. 1.

Molecular serotype identification based on the sequence heterogeneity of the 790-bp fragment (positions 1437 to 2226, corresponding to the regions of the amplicon obtained with primer pair cpsES3-cpsGA1) at the 3′ end of cpsE-cpsF and the 5′ end of cpsG (the relevant primers are indicated). Numbering start point 1 refers to start point 1 of the sequence with GenBank accession number AF332908 (for serotype reference strain 3139). Dashes indicate the same sequence as the consensus sequence; *, for serotype Ib, position 2221 refers to the sequence with GenBank accession number AB050723.