Identification of Group B Streptococcus Capsule Type by Use of a Dual Phenotypic/Genotypic Assay

Abstract

The group B streptococcus (GBS) capsular polysaccharide (CPS) is an important virulence factor which is also used for GBS typing. There are 10 CPS types (Ia, Ib, and II to IX). GBS that do not phenotypically type are considered nontypeable. All genes required for CPS synthesis are found on the GBS cps operon, which contains a highly variable CPS-determining region (cpsG-cpsK). The objective of this study was development of an assay to detect sialic acid on the GBS cell surface, followed by a genotypic PCR CPS typing assay. Sialic acid is located at the terminal end of the side chain of all known GBS CPS types. Sialic acid can be bound to commercially available lectins such as slug Limax flavus lectin. Biotinylated L. flavusstreptavidin-peroxidase complex was used in an enzyme immunoassay and dot blot assay to detect sialic acid. This was followed by a PCR typing scheme that was developed to target the serotype-determining region of the cps locus for Ia, Ib, and II to IX. Sialic acid from the CPS types Ia, Ib, and II to IX was detectable on the GBS cell surfaces of all previously identified CPS-typed GBS strains assayed. This was followed by the real-time PCR typing assay which successfully identified CPS Ia, Ib, and II to IX types. The combination of phenotypic and genotypic assays provides an accurate tool for detection of CPS expression and assignment of CPS typing. These assays have the potential to be used for CPS typing in large-scale epidemiological studies.

Phenotypic and molecular analysis of nontypeable Group B streptococci: identification of cps2a and hybrid cps2a/cps5 Group B streptococcal capsule gene clusters

“…Knowing that the presence of surface sialic acid can predict the presence of polysaccharide capsule, it was unusual to find in our collection three GBS isolates (PLGBS16, PLGBS17, and PLGBS18) which failed to react with antisera raised against the nine known CPS types (Ia, Ib, and II–VIII) yet were sialic acid positive 23 (data not shown). Additionally, these three isolates could not be genotyped by our previously described RT-PCR typing assay (which includes Ia, Ib, and II–IX) 23 , suggesting novel mechanisms of encapsulation. A polysaccharide stain revealed that these three GBS isolates microscopically displayed CPS surrounding the cell wall (Supplemental Figure 3 ).…”

“…The relevant characteristics of the bacterial strains used in this study are listed in Table 4 . We reported previously that 159/1683 GBS isolates collected from patients between 2003 and 2013 were identified as NT 31 and of those, 84 of the 159 were sialic acid negative (sia(−)) 23 , whereas 75/159 were sialic acid positive (sia(+)) 23 . Of the 75 serologically nontypable sia(+) isolates, only three isolates (PLGBS16, PLGBS17, and PLGBS18) could not be genotyped by our previously described RT-PCR typing assay (which included Ia, Ib, and II–IX) 23 .…”

“…All recognized GBS CPS types have sialic acid attached to their CPS structure 17 – 20 that can interfere with complement-mediated killing by host immune cells 21 , 22 . Based on the conservation of sialic acid among all recognized GBS CPS types and the essentiality of its presence for full capsule biosynthesis and expression 17 , 18 , sialic acid has been utilized as recognition marker for GBS CPS production 23 .…”

“…We reported previously that 9% of GBS isolates in Alberta, Canada, collected from patients with invasive diseases between 2003 and 2013 were identified as NT 31 and of those, 52.8% were sialic acid negative (sia(−)) 23 . The sia(−) phenotype in NT GBS isolates suggests loss of cps expression as sialic acid is present on all known GBS CPS types.…”

“…This suggested that the NT GBS isolates that were sia(−) in our collection may have genetic mutations in the covR/S genes resulting in a loss of CPS expression 25 , 27 , 28 . We also previously identified from this collection three sialic positive (sia(+)) GBS isolates that could not be CPS typed by a serological assay or by PCR 23 .…”