Development of a Pathogen Profiling Approach for Detecting and Dissecting Markers of Pathogenicity and Hyper-Variability in Group B Streptococci

Loy, Richard Paul (2013). Development of a Pathogen Profiling Approach for Detecting and Dissecting Markers of Pathogenicity and Hyper-Variability in Group B Streptococci. PhD thesis The Open University.



Sequence typing is a rapidly evolving field and offers improved analysis into the genetic background and lineages of organisms compared to serological or DNA banding pattern based analysis. However, the resolution of molecular typing schemes varies between organisms and often loci used in sequence typing lack discriminatory power and give limited information into the evolution of the organism. This is particularly true of group B streptococci (GBS), where the same sequence types appear worldwide, which is unlikely for such a pathogen.

This project aimed to develop a two component pathogen profiling approach which accurately reflected the phylogeny of GBS isolates, using elements of the core genome and elements of the variable genome. To address this a bioinformatic approach which selected loci for sequence typing based on predicting genes which evolve in the same manner as the average for the core genomes was adapted from a previously applied study for designing genus level sequence typing schemes. This informed the selection of candidate loci which were then experimentally verified, using a collection of 135 GBS clinical isolates. It was demonstrated that it was possible to obtain greater resolution and accuracy using only three unique genes that are intelligently selected, rather than using seven known housekeeping genes that are selected at random.

Sources of hyper variability within the genome, in particular the presence of mononucleotide repeats (MNR) were investigated in non-coding DNA. It was postulated that these regions of DNA are more prone to mutation due to the lack of selective pressures, the presence of MNR repeats make these regions more unstable during replication and that in core genes these regions may be involved in genomic regulation by slipped strand mispairing. Results did confirm that non-coding DNA containing MNR repeats were more variable than DNA without them but these did not match the discriminatory power of MLST typing or the new three gene typing scheme. However, it was observed that one MNR tract was an insertion site for one of two insertion sequences and that typing using the presence/absence of these insertion sequences further enhanced discriminatory power in addition to the 3 gene scheme and may yet prove to be an indicator of virulence in clinical GBS isolates.

As well as demonstrating that sequence typing can be more informative if sequence typing markers are intelligently selected, this project also showed the importance of developing computational methods to analyse pathogen genome sequences that are being released in ever increasing numbers thanks to new and constantly improving technologies. For example, methods-used here to determine the core and pan-genome of any given set of genomes are becoming increasingly important to the study of pathogen evolution, virulence and population dynamics.

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