Molecular Characterization of Streptococcus agalactiae Isolates from Human Cases of Invasive Disease Collected in Alberta, Canada: 2003-2013

• Author / Creator

  Alhhazmi, Areej

• Capsular polysaccharide (CPS) surrounding the Group B streptococcus (GBS) is a major virulence factor for GBS. There are 10 recognized CPS types (Ia, Ib and II-IX). A GBS isolate is non-typeable (NT) when CPS cannot be identified as one of the 10 CPS types. All genes required for CPS synthesis are found on the GBS cps operon, which contains a highly variable CPS-determining region (cpsG-cpsK). Sialic acid (sia) is located at the terminal end of the side chain of all known GBS CPS types. In GBS, CovRS is an important regulatory system that controls GBS virulence factors, including the capsule.
  Invasive GBS (iGBS) from Alberta, Canada between 2003-2013 were analyzed to determine prevalence rates of GBS disease, CPS type distribution, and antimicrobial susceptibility patterns. Over the 11 years, a noticeable increase in the rate of neonatal GBS disease as well as adult infections was documented including CPS Ia, Ib, III, and IV. The increased rates of iGBS disease have been accompanied by elevated rates of erythromycin and clindamycin resistance.
  A comprehensive CPS typing system was developed to detect sialic acid on the GBS cell surface followed by a genotypic PCR CPS typing assay. Sialic acid can be bound to commercially available lectins such as slug Limax flavus (LFA) lectin. Biotinylated LFA-streptavidin-peroxidase complex was used in EIA and dot blot assays to detect sialic acid. That was followed by a PCR typing scheme targeting the serotype-determining region of the cps locus for Ia, Ib, and II-IX. The combination of sialo-CPS lectin binding and duplex real-time PCR typing assays provided a simple and reliable tool for CPS expression confirmation and CPS genotype. This system enabled the characterization of GBS CPS Ia, Ib, II-IX, thereby reducing the rate of detection of NT isolates. Two groups of GBS NT isolates were studied, isolates without surface sialic acid sia (-) and isolates with surface sialic acid sia (+). NT sia (-) isolates were characterized by assaying in vitro virulence changes and identifying covR/S mutations potentially responsible for altered virulence phenotypes. NT sia (+) isolates were investigated to identify genetic changes in the cps operon that failed to identify as one of the 10 CPS types yet expressed capsule. A subset of non-capsule producing and sia (-) isolates identified a series of covR/S mutations not only affecting CPS production but also an array of other phenotypic properties. Of the NT CPS expressing strains and sia (+), two isolates were identified as a subtype CPSIIa encoded by CPSIIa gene operon. One isolate was found to encode a novel CPS type, CPSIIa/V hybrid. Whole genome sequence (WGS) technologies elucidated the complete genome of a ST 1/serotype V PLGBS13 strain recovered from an adult case in Alberta, Canada in 1997. Polymorphism and recombinational studies from four iGBS ST1 isolates revealed that the genetic diversity among ST 1/serotype V GBS isolates is mainly driven by small genetic changes such as insertions, deletions or mutations. However, the genetic diversity of ST 1 GBS isolates that have serotypes other than serotype V (CPS type IIa and cpsIIa and V hybrid) is mainly explained by recombination in the cps gene cluster and few genes.
  In conclusion, the research presented in this thesis revealed that Alberta has experienced an increase in the incidence of GBS disease. A dual GBS typing system involving phenotypic and genotypic assays was developed. These assays were reliable, sensitive and specific and enable the characterization of GBS CPS Ia, Ib, II-IX, thereby reducing the rate of detection of NT isolates. A series of covRS mutations among NT sia (-) isolates was identified. Of the NT CPS expressing strains, two subtypes of CPSII do exist, CPSIIa and CPSIIb each encoded by cpsIIa and cpsIIb gene operons, respectively. Sia (+) detection assay allows for the discovery of novel CPS producing strains such as the CPSIIa/V hybrid. This work also contributed to the understanding of the genetic diversity among ST1 isolates commonly found among adult population.

• Subjects / Keywords

  • Polymorphism
  • Whole genome sequence
  • Lectins
  • Genotype
  • GBS
  • Capsular polysaccharide
  • Typing
  • covRS mutations
  • Sialic acid

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3SJ1B692

• License

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