Application of whole genome sequencing to characterize Mycobacterium tuberculosis strains circulating in Far North Queensland, Australia and Western Province, Papua New Guinea

Abstract

Mycobacterium tuberculosis (MTB) is an obligate intracellular human pathogen that causes tuberculosis (TB) disease. Today, TB is the leading cause of death from a single infectious agent and ninth leading cause of death globally. Increasing burden of drug-resistant TB is threatening the achievements and targets set by World Health Organisation (WHO). With the recent advances in whole genome sequencing (WGS) technology, there is renewed hope for its role as a ‘game changer’ towards eliminating TB. This thesis focusses on the utility of WGS technology on MTB strains circulating in Far North Queensland (Australia) and Western Province (Papua New Guinea) for extensive understanding of molecular epidemiology and evolutionary dynamics that are important for improved TB management. Western Province, Papua New Guinea (PNG) which neighbours Far North Queensland has one of the highest numbers of drug resistant TB globally. Various efforts are being made to control the epidemic spread of TB within this region and the surrounding areas.In the first research study (chapter 2), isolates from Daru (capital town, Western Province) were first analysed using a conventional genotyping method (MIRU-VNTR) which identified high level of strain relatedness indicative of ongoing transmission. On application of WGS, four different clades of a dominant modern Beijing strain were identified. Molecular markers that contribute to development of drug resistance, virulence and transmission were characterized. Molecular dating of the dominant strain suggested a long evolutionary history, estimated to have started in the 1940s and early acquisition of streptomycin and isoniazid resistance markers in the 1950s. Some of the identified genomic markers within the Beijing strain highlight possession of plausible biological mechanisms that contribute to the on-going transmission of drug resistance in the study setting and threaten further spread to other regional areas.In the second study (chapter 3), both genomic and epidemiological data were exploited to understand the diversity of strains circulating among PNG nationals (cross-border patients) who sought TB services at Australian clinics within the Torres Strait. This is part of understanding the prevailing transmission dynamics within the Torres Strait Protected Zone (TSPZ). This zone has free movement of people (no visas) between Australia and PNG territories. Modern Beijing strain was identified to be the most prevalent strain, and it was significantly associated to transmission especially among patients from the same locality. As ‘proof of principle’ for cross-border transmission among nationals of either countries in TSPZ, four epidemiologically linked Australians isolates (within the Torres Strait) were characterized to have attained the same a drug resistant Beijing strain from PNG. This highlighted the need for enhanced co-operation among regional authorities for improved surveillance of TB and the necessity for integration of WGS into the regional surveillance program.It should be noted that during genomic analysis of the two above mentioned research studies, variants within genomic regions such as proline-glutamate/proline-proline-glutamate (PE/PPE) gene families were consistently filtered out as they were considered spurious. These regions have a rich GC content and repetitive in nature which makes it difficult to study using short-read sequencing data like Illumina. Moreover, the diversity within these genes is thought to contribute to the diverse pathogenicity, immunogenicity and virulence among MTB strains. So in the third study (chapter four), Oxford nanopore technologies (ONT) which is known to produce long reads and high depth sequence data was utilized on an extensively drug resistant (XDR) strain as proof of principle for its applicability. This technology did not only provide unparalleled resolution to assemble the first circular XDR genome that included the PE/PPE genes, it also unravelled novel variations within these regions hence highlighting some of the mechanisms played by these genes towards immunogenicity and virulence. Unique structural variants were identified from the assembled genome and it has potential to serve as a local reference genome for future MDR/XDR surveillance.In conclusion, WGS provided greater insights into the molecular basis of resistance, evolution and transmission dynamics of strains circulating in Western province, PNG, Torres Strait and North Queensland. Future assessment of dominant modern Beijing strains’ extent of spread to wider areas of PNG and north Queensland will be required. Analysis performed indicated significant clinical and public health benefit towards strengthening TB surveillance programs and improve TB patient management. ONT demonstrated its usefulness in providing further insights into the genetics of an XDR MTB strain, though further evaluation of its use is needed in high burdened settings.