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Burkholderia pseudomallei in northern Australia : sequencing approaches to better biogeographical and epidemiological understanding

McRobb, Evan T. (2015). Burkholderia pseudomallei in northern Australia : sequencing approaches to better biogeographical and epidemiological understanding. PhD Thesis, Charles Darwin University.

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Author McRobb, Evan T.
Title Burkholderia pseudomallei in northern Australia : sequencing approaches to better biogeographical and epidemiological understanding
Institution Charles Darwin University
Publication Date 2015-11
Thesis Type PhD
Supervisor Currie, Bart J.
Abstract This research embodies conventional and next generation genetic analysis of the causative agent of Melioidosis, the gram-negative bacterium Burkholderia pseudomallei. Melioidosis is a disease of public health importance for hyperendemic areas in northern Australia and Southeast Asia, where B. pseudomallei is ubiquitous in the environment. This disease is contracted when individuals are exposed to soil or water containing the bacterium, which typically enters the body via abrasions in the skin, inhalation, or ingestion. Despite 100 years of research since the discovery of this bacterium, knowledge gaps remain; particularly surrounding B. pseudomallei strain diversity in the environment, and as a result, what ramifications extend to human melioidosis cases.

The Northern Territory, Australia, has the highest incidence of melioidosis globally (50.2 cases per 100,000); in the first research chapter we aim to explore the biogeography of B. pseudomallei in this region, i.e. the dispersal of this bacterium in the environment. Multilocus sequence typing (MLST) was performed on a large dataset of (n=953) B. pseudomallei isolates collected over the past two and a half decades in the Northern Territory, and the adjacent state Queensland. These isolates originated from both humans and environmental samples, and were assigned geographic regions of origin from detailed collection data. We generated diversity indices (Simpson’s index and evenness) and compared them between these regions using MLST data. B. pseudomallei strains diversified as our geographic sampling area increased. In addition, we found only few dominant STs in smaller sampling regions, and the inverse in larger areas, suggesting that strains radially expand throughout the environment over time. Further, the environmental and clinical B. pseudomallei isolates didn’t have any ST bias, suggesting environmental strains are all potentially invasive.

Case clusters of melioidosis have been previously noted in the Northern Territory, chiefly underground water systems that are used for domestic water supplies. In 2012, two melioidosis cases over a three-month period were suspected of originating from a water supply in the rural Darwin region. We aimed to investigate this property by sampling soil surrounding the dwelling and the water supply. Quantitative culture of B. pseudomallei was performed to ascertain the levels of contamination if the bacterium was present. B. pseudomallei was isolated from both soil and the water supply, whereas high concentrations of the bacterium were found in the water supply (500CFU/mL). Clinical and environmental samples were subjected to MLST, which found the two cases, and all water supply B. pseudomallei isolates were ST325. Soil isolates were a different ST and therefore disregarded as the probable infecting source. As a measure to protect the occupants in the household, and to inform strategies to protect occupants in future high-risk households, a UV filter was installed in the water supply. The UV filter was found to be effective, reducing B. pseudomallei contamination to undetectable levels using both culture and sensitive Real-Time PCR.

B. pseudomallei has recently been classed a Tier 1 select agent, the highest ranking for a biological agent, and shares the category with the Ebola virus, Bacillus anthracis (Anthrax), and Yersina pestis (Plague). There are fears that microorganisms in this category may be used as a bioweapon. The ability to trace deliberately released pathogens has been facilitated by an advance in genome sequencing technology for forensic purposes. A melioidosis case has never been linked to an environmental source using these genomic tools. In this final research chapter we aimed to describe an environmental B. pseudomallei strain that likely infected two occupants of the household detailed in the previous chapter. Whole genome sequencing was applied to B. pseudomallei strains from the two occupants and their water supply, and also unrelated strains from human cases and water supplies in the nearby area that served as out-groups to validate our findings. We also used a novel approach of analyzing whole genome data; combining single nucleotide polymorphisms (SNPs) with insertion/deletions (indels). Phylogenetic analysis of the genomes revealed a probable
infecting strain in the occupant’s water supply. SNP and indel data were concordant, and accurately described the epidemiological events, more so than using each analysis separately. These results are important for informing attempts to trace outbreaks of melioidosis, and contribute to the forensic tracing tools available for tracing organisms of biosecurity importance.

These three chapters describe the use of conventional, and next generation DNA sequencing technology to study B. pseudomallei in the hyper-endemic region northern Australia. We have explored the biogeography of this bacterium finding high levels of diversity, and evidence to suggest strains radially spread in the environment over time. In the Darwin region of the Northern Territory, we have responded to a human case cluster, finding high concentrations of B. pseudomallei in the household water supply. A UV filtration system was trialed and found to effectively decontaminate the water supply, a cost effective strategy to prevent melioidosis cases in this region. Furthermore the probable infecting environmental B. pseudomallei strain was identified for the first time in a human melioidosis case. A novel approach was used combining SNP and indel data, an important addition to the genotyping tools used for pathogens of biodefense interest. These data contribute to our knowledge of B. pseudomallei in the environment of a hyper-endemic region, means to decontaminate water supplies that pose a risk to human health, and guide efforts to trace strains that
may be used for nefarious purposes.
Keyword Burkholderia pseudomallei
Burkholderia mallei
Burkholderia thailandensis
single nucleotide polymorphism
whole genome sequencing
Multilocus sequence typing
Multilocus VNTR analysis

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