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  • (2020) Khan, Mahjabeen
    Thesis
    Pseudomonas aeruginosa causes both contact lens and non-contact lens-related keratitis (corneal infection). This opportunistic bacterium naturally has the ability to resist the mechanism of action of many antibiotics which are used for treatment. P. aeruginosa resistance patterns and the mechanism of resistance in isolates from keratitis are not well understood. This thesis described the phenotypic and genotypic patterns of antimicrobial resistance and compared these between ocular isolates of P. aeruginosa from Australia (contact lens) and India (non-contact lens). Changes in the antimicrobial susceptibility between isolates over time were also analysed. Susceptibility to antibiotics, multipurpose disinfecting solutions and disinfectants was analysed for twenty-seven Australian isolates from contact lens-related keratitis and forty non-contact lens-related isolated from India. The whole genomes of fourteen Australian (historical and recent) and twelve Indian isolates were sequenced using Illumina® MiSeq®. Computational analysis of the genomes was performed to analyse their core and pan genomes and these were examined for the presence of acquired resistance genes, virulence genes, gene mutations, and these compared to their phenotypic resistance to antibiotics. Indian isolates possessed large pan genomes with more acquired resistance (30) genes and larger numbers of genetic variations. The Indian isolates contained clones of three sequence types ST308, ST316 and ST491, whereas Australian isolates contained only one sequence type ST233. Isolates with larger gene variations had mutations in the DNA mismatch repair system. Most multi-drug resistant Indian (non-contact lens) isolates were exoU +. Indian isolates had large accessory genes compared to Australian isolates and this increased the pan genome size of the Indian isolates. The number of core genome mutations were larger in the Indian isolates a median of 50006 (IQR=26967-50600) compared to Australian isolates a median of 26317 (IQR=25681-33780). There were differences between isolates from Australia and India with respect to their antibiotic resistance and associated genes. Indian strains had more genetic diversity and were multi-drug resistant. However, there was no evidence of substantial genetic or phenotypic changes within isolates from their respective countries.

  • (2024) Rayamajhee, Binod
    Thesis
    Acanthamoeba, a free-living heterotrophic protist, typically resides in a variety of aquatic environments. It is known to pose a risk to humans, causing various diseases, including eye infections (keratitis), often transmitted through contact lenses. The primary risk factor for Acanthamoeba keratitis (AK) in developed countries is engaging in recreational water activities while wearing contact lenses. Late and misdiagnosis of AK are commonly observed, often leading to worse clinical outcomes. Treatment of Acanthamoeba infections is challenging due to the absence of drugs that can effectively eradicate both trophozoites and resilient cysts. In a remarkable dual role, Acanthamoeba behaves as a phagocytic predator, consuming other microbes, but also as an environmental host for diverse bacteria which can resist the intracellular killing mechanism of the amoebal host. Among AK patients, coinfections with various bacteria and fungi have been reported in recent years, presenting as a complex polymicrobial keratitis. The research in this thesis investigates the incidence of Acanthamoeba in Sydney's largest domestic water reservoir and determines the associations between Acanthamoeba and physicochemical as well as microbiological parameters of the water. In addition, the research evaluated the incidence of Acanthamoeba spp. within coastal lagoons on the eastern coast of Australia using a metagenomic approach and investigate their associations with aquatic bacteria. The research also examined the genotypes of Acanthamoeba species recovered from AK patients in Hyderabad, India and Sydney, Australia, along with the types of intracellular bacteria they harbor, using microbiome sequencing. Finally, the research examined whether naturally acquired intracellular bacteria in Acanthamoeba led to more severe ocular infections in an animal model. In this thesis, two review papers (chapters 1B and 1C) summarised the growing importance of Acanthamoeba as a potential human pathogen, elucidating its role as an environmental predator. An invited narrative review (chapter 1B) highlighted how Acanthamoeba predation in environmental habitats can be a key factor shaping the structure and composition of microbial populations. This review comprehensively examined and listed Acanthamoeba-resisting bacteria, along with in vitro interactions and adaptations between Acanthamoeba and bacteria. A systematic review in this thesis (chapter 1C) examined the diversity of microbial species identified in environmental and clinical isolates of Acanthamoeba spp. The comprehensive systematic review also outlined the knowledge gap of whether Acanthamoeba minimicrobiome contains phylogenetically distinct bacteria. In the past, approximately 20% of AK patients in the Sydney metropolitan reported swimming in seawater or freshwater while wearing their contact lenses before they became infected. The current research identified that 34.5% of water samples from Warragamba dam, the largest water reservoir in Sydney contained viable Acanthamoeba spp. (chapter 2), and the incidence of Acanthamoeba spp. in the dam was positively correlated with a higher concentration of algal biovolume. Additionally, 38% of water samples from four lagoons on the NSW, Central Coast were positive for Acanthamoeba, with significantly higher numbers observed in summer compared to other seasons (chapter 3). Water from a highly urbanised area were more commonly infested with Acanthamoeba than non-urbanised areas. Most strains belonged to the pathogenic genotype T4 clade. Water turbidity, temperature, intl1 gene concentration (a proxy measure of anthropogenic pollution), and dissolved oxygen were significantly associated with Acanthamoeba numbers. This study (chapter 3) found a positive correlation between the abundance of Acanthamoeba in coastal waterways and the levels of cyanobacteria, Pseudomonas spp., Candidatus Planktoluna, Curvibacter sp., Polynucleobacter cosmopolitanus, Mycobacterium spp., Vibrio pacinii, and marine bacteria of the Bacteroidota phylum. Overall, the presence of Acanthamoeba spp. had a significant influence on the composition of bacterial communities in lagoons (chapter 3). Subsequent studies in this thesis investigated the genotypes of Acanthamoeba recovered from ocular infections in Hyderabad (chapter 4A) and Sydney (chapter 4B). Most Indian (92.3%) and all Australian corneal isolates belonged to genotype T4. Among Indian isolates, nine strains harboured intracellular bacteria and one contained the fungus Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates, epithelial defect and hypopyon compared to AK cases without intracellular microbes. Ocular trauma was the major reported risk factor among AK patients in Hyderabad, India where 46.2% required surgical intervention, and one case underwent evisceration. This study also analysed domestic tap water of nine AK patients in Sydney, and Acanthamoeba was detected in four of them. Notably, one isolate was found to harbor viable Pseudomonas aeruginosa (chapter 4B). Chapter 5 of this thesis assessed the intracellular microbiome composition of 51 Acanthamoeba species isolated in Australia and India, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. Distinct microbial abundance patterns were observed among the samples; Pseudomonas species were abundant in Australian corneal isolates, Enterobacteriales in Indian ocular isolates, and Bacteroidota was abundant in Australian water isolates. More diverse intracellular bacteria were identified in water isolates as compared to clinical isolates. The beta diversity measure of bacterial communities in corneal isolates of Acanthamoeba exhibited significant difference based on the country of origin, while alpha diversity did not vary significantly as per the source of isolation or country of origin. Chapter 6 of this thesis examined the impact of naturally acquired viable intracellular P. aeruginosa in the development of AK in rats cornea. The findings from this study (chapter 6) demonstrated that AK infection in rat's corneas, using an Acanthamoeba strain containing intracellular P. aeruginosa, resulted in acute keratitis with a high neutrophil response, whereas Acanthamoeba alone induced only very mild infections. During the infection in rat corneas, intracellular P. aeruginosa cells were expelled, as no intracellular bacteria were observed in Acanthamoeba trophozoites reisolated from the rats' corneas. No severe keratitis developed when Acanthamoeba and intracellular P. aeruginosa were separately inoculated in distinct groups of rat corneas, unlike their combined inoculation. These findings suggest that the concurrent infection of Acanthamoeba, coupled with the release of intracellular bacteria onto a compromised cornea, can contribute to the development of complex microbial keratitis. In summary, this thesis observed a relatively high prevalence of pathogenic Acanthamoeba in both the water reservoir and coastal waterways of Sydney. It also identified potential contributors in water habitats that could promote the proliferation of Acanthamoeba strains, determined circulating genotypes of Acanthamoeba among AK patients in Hyderabad and Sydney, comprehensively characterised the microbiome that can inhabit Acanthamoeba spp., and assessed the impact of a member of this microbiome on corneal infection in rats.