The clinical application of whole-genome sequencing to the surveillance, understanding, and control of bacterial infectious diseases in Australia

© 2017 Dr. Jason Chek-Hoi KwongThe scientific advance of whole-genome sequencing (WGS) has revolutionised the field of microbiology, with the ability to examine, characterise and compare organisms at a DNA sequence level. In recent years, the improving costs and workflows to sequence bacteria have resulted in WGS emerging as a powerful technology with the potential to enhance clinical and public health microbiology. However, at the commencement of this PhD, experience in clinical and public health institutions was limited, with only a few small-scale proof-of-concept applications in Australia. This thesis explores three different applications of WGS in a public health microbiology reference laboratory. For national surveillance of Listeria monocytogenes, a foodborne pathogen of public health significance, WGS provided superior resolution to current typing methods used to affirm potential outbreaks of Listeria. Additionally, typing results could be predicted in silico from the WGS data for backwards compatibility. The implementation of routine, prospective WGS in a national surveillance system for Listeria was feasible, though three important issues for future implementation were identified. Firstly, for comparing genomes of the same strain type, the use of a closely related reference genome produced a more accurate and higher resolution phylogeny than using a distantly related reference genome. Secondly, WGS identified nucleotide-level differences between isolates previously assumed to be "isogenic". Thirdly, the optimal method to define an outbreak with the genomic data was unclear. These issues were further explored in using WGS to understand sexual transmission of Neisseria gonorrhoeae between men who have sex with men (MSM), and the increasing rate of antimicrobial resistance (AMR) among Neisseria gonorrhoeae isolates. For 33 of 34 MSM couples infected with gonorrhoea, the same strain of Neisseria gonorrhoeae was found in both partners, and within individual MSM infected at multiple anatomical sites. However, subtle nucleotide differences were again observed between "within-host" and "within-partnership" isolates. Genetic determinants of AMR were able to be identified from the WGS data, with the absence of differences between within-host and within-partnership isolates suggesting that increasing AMR among gonococcal isolates is driven by transmission from sexual partners, and controlling transmission of gonorrhoea is critical to limiting the spread of AMR. Following this, the use of real-time WGS was investigated to assist with the state-wide control of another group of resistant pathogens, KPC-producing Enterobacteriaceae, in Victoria, Australia. Due to the complexity of the outbreak, combined genomic and epidemiological analyses provided greater insight into the outbreak than the data from each approach separately. These analyses defined distinct transmission networks arising from multiple importations into Victoria in separate healthcare facilities, allowing more targeted public health interventions. Additional insights into the outbreak were gained through long-read sequencing to track interspecies plasmid transfer, multiple colony selection for sequencing to capture within-host diversity, and Bayesian evolutionary and transmission modelling to estimate the transmission tree, informing public health responses to the outbreak. These studies provide a framework for the implementation of WGS in public health microbiology in Australia, with the tools developed and datasets derived through this work adding to the resources available to assist future clinical applications of WGS