Diversity and bioactivity of microorganisms associated with Australian stingless bee species

Abstract

The rapid emergence and transfer of antimicrobial resistance in pathogenic organisms has greatly reduced our ability to treat clinical microbial infections. To continue to treat clinical microbial infections in humans and animals it is imperative that we discover new antimicrobial compounds with novel modes of action. The aim of this thesis was to explore the antimicrobial potential of microorganisms associated with three Australian native stingless bees, Tetragonula carbonaria, Austroplebeia australis and Tetragonula hockingsii. Observations of antimicrobial activity from honey, hive materials and whole extracts of Australian stingless bees inspired the hypothesis that the microbiota associated with these stingless bee species could produce bioactive compounds. To address this aim, a comprehensive evaluation of the native bee whole gut microbiome was performed (Chapter 2), with the goal of identifying microorganisms exhibiting specific associations. Guided by these results bacteria and fungi were cultured from the gut and cuticle of three species of Australian stingless bees and screened by genetic and chemical methods to create a subset enriched for chemical diversity and biosynthetic potential (Chapter 3). Biosynthetic potential was correlated to the presence of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) gene clusters due to their historical success as therapeutic natural products and their potential to produce a wide array of bioactive chemical structures. Antimicrobial activity of the selected microbes was assessed by liquid culture bioassay and a successful candidate identified for bioactivity-guided fractionation and compound characterisation (Chapter 4). The antimicrobial long chain fatty acid, 9-hexadecenoic acid, was isolated and characterised from the Xanthomonas sp., TMB - 122. Additional characterisation identified the production of 2,5-di-tert-butylphenol, a phenolic compound with antimicrobial activity, and tridec-1-ene, an acyclic olefin insect pheromone, by Xanthomonas sp., TMB - 122. This investigation confirmed our hypothesis that microbes associated with Australian stingless bees were bioactive. Furthermore the combined genetic and chemical analyses performed validated the selection of candidates possessing PKS and NRPS gene clusters as this methodology accounted for 75% of the chemical diversity observed by LC-MS profiling. The steps enclosed in this thesis are the practical application of the theory that modern drug discovery methods must build on the knowledge off the past whilst continuing to innovate and explore.