Development of diagnostic tools for the detection of Streptococcus agalactiae

Abstract

Streptococcus agalactiae is a common opportunistic pathogen, which, when transmitted to the immunocompromised, elderly or newborns, can cause complications such as sepsis, pneumonia, meningitis and can also be fatal. In screening programmes expectant mothers are typically sampled at 35-37 weeks gestation to ensure microbial culture results are available prior to labour. As levels of S. agalactiae rapidly fluctuate, premature testing results in antibiotic prescription being informed by outdated information. This project aimed to develop tools for use in a sensitive, specific, point-of-care diagnostic test able to detect all strains of S. agalactiae. Three detection strategies were pursued based on peptide aptamers, antibodies and DNA amplification. A bioluminescent protein, aequorin, was selected for use as a peptide aptamer scaffold in order to link epitope detection to reporter signal. Peptide insertion into aequorin eliminated expression of soluble protein, and therefore was deemed unsuitable for aptamer library construction. A novel computer programme, IDRIS, identified peptides conserved throughout all currently sequenced S. agalactiae strains, which are also unique to the species. Through further bioinformatic analysis, three peptide sequences from SAG1474 protein were selected for in-house murine monoclonal antibody production. Antibodies targeted against one peptide (CVNLEENSQV) exhibited sensitivity to all S. agalactiae serotypes. Antibodies exhibited species specificity when tested against Streptococcus pyogenes; future work will focus on extending specificity studies, including analysis of bacteria present in the vaginal flora. Recombinase polymerase amplification is a rapid, isothermal DNA amplification technique that may enable inexpensive point-of-care testing. Target-specific primers with single stranded overhang sequences allow amplified product to hybridise to a complementary capture probe and reporter-tagged detection probe. The plate-based system can detect 3.8 x 104 copies of DNA and is able to specifically detect all S. agalactiae serotypes over S. pyogenes DNA. The test will be converted into a lateral flow assay in the future