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Development of Diagnostic Tools for the Detection of Streptococcus Agalactiae

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Development of Diagnostic Tools for the Detection of Streptococcus Agalactiae Development of Diagnostic Tools for the Detection of Streptococcus agalactiae Hannah Swinburne Thesis submitted for the degree of Doctor of Philosophy Institute of Cellular Medicine Faculty of Medical Sciences Newcastle University November 2017 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 As levels of S. agalactiae rapidly fluctuate, premature testing results in antibiotic prescription being informed by outdated information.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. i Declaration I hereby declare that this thesis has been composed by myself and has not been submitted in any previous application for a degree. The work presented has been performed by myself, unless otherwise stated. All sources of information have been appropriately acknowledged by means of reference. ii Acknowledgements I am grateful to the EPSRC for funding this work. I would like to thank Neil Keegan, Chris Johnson and Calum McNeil for their continual support throughout the project. Their knowledge, help, ideas, guidance and encouragement have pushed me to become a better scientist. Thanks to Julia Spoors and Rob Bolt for countless reasons, most significantly in all their work in antibody development. Thanks to Beth Lawry for her advice in every aspect of the project. I’d like to thank Keith Flanagan, Elisa Anastasi and Anil Wipat for their help using the IDRIS system, Helen Glenwright and Wendy Smith for their support whilst working in the Centre for Bacterial Cell Biology and Nicholas Jakubovics for kindly donating S. pyogenes strains. Special thanks to Cara, Emily, Gina, Katie, Suzie, Carl, Phil, Chen and all those mentioned above for their friendship; it has made the past three years wonderful. iii Contents Chapter 1 Streptococcus agalactiae: Bacteria, Disease and Diagnostics .................. 1 1.1 Streptococcus agalactiae bacterium .................................................................. 1 1.1.1 Streptococcus agalactiae ............................................................................ 1 1.1.2 Serotypes .................................................................................................... 3 1.1.3 Related bacteria belonging to the genus streptococcus .............................. 3 1.1.4 Virulence factors.......................................................................................... 6 1.2 Group B streptococcal disease .......................................................................... 8 1.2.1 Early onset disease ..................................................................................... 8 1.2.2 Late onset disease .................................................................................... 10 1.2.3 Immunocompromised ................................................................................ 11 1.2.4 Elderly ....................................................................................................... 12 1.2.5 Worldwide disease .................................................................................... 13 1.3 Treating and preventing GBS disease ............................................................. 13 1.3.1 GBS vaccine development ........................................................................ 13 1.3.2 Antibiotic treatment .................................................................................... 14 1.3.3 Maternal screening/risk- based treatment ................................................. 16 1.4 Diagnosis of S. agalactiae infection ................................................................. 18 1.4.1 Current GBS diagnostic tests .................................................................... 18 1.4.2 Rapid test .................................................................................................. 24 1.5 Project aims ..................................................................................................... 26 Chapter 2 Methods ................................................................................................... 28 2.1 Materials .......................................................................................................... 28 2.2 General techniques .......................................................................................... 29 2.2.1 Bacterial cell growth .................................................................................. 29 2.2.2 Bacterial cell storage ................................................................................. 30 2.2.3 SDS-PAGE ................................................................................................ 30 iv 2.2.4 Western blotting ........................................................................................ 31 2.2.5 Indirect enzyme linked immunosorbent assay (ELISA) ............................. 32 2.2.6 Protein concentration determination – BCA assay .................................... 33 2.2.7 Protein concentration determination – spectrophotometry ........................ 33 2.2.8 RNA extraction .......................................................................................... 33 2.2.9 cDNA synthesis ......................................................................................... 33 2.2.10 DNA extraction ........................................................................................ 34 2.2.11 Polymerase chain reaction ...................................................................... 34 2.2.12 Agarose gel ............................................................................................. 34 2.2.13 Nucleic acid concentration determination - spectrophotometry ............... 35 2.2.14 DNA purification ...................................................................................... 35 2.2.15 Statistics .................................................................................................. 35 2.3 Computing ....................................................................................................... 36 2.3.1 IDRIS ......................................................................................................... 36 2.3.2 PSORTb .................................................................................................... 37 2.3.3 SignalP ...................................................................................................... 37 2.3.4 LipoP ......................................................................................................... 37 2.3.5 TMHMM .................................................................................................... 38 2.3.6 I-TASSER .................................................................................................. 38 2.3.7 PSIPRED .................................................................................................. 38 2.3.8 Prodepth .................................................................................................... 39 2.3.9 Bcepred ..................................................................................................... 39 2.3.10 Microarray data analysis ......................................................................... 39 2.3.11 Protein BLAST (Basic local alignment tool) search ................................. 40 2.3.12 DNA alignment ........................................................................................ 40 2.3.13 Primer design: PCR and RPA ................................................................. 40 2.3.14 Band densitometry .................................................................................. 41 v 2.4 Recombinant protein
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