Leveraging Capillary-Assembly to Design Microbial Communities

Abstract

Bacteria typically live in dense, surface-attached, biofilm-embedded communities in close proximity and competition with other microbes. Yet despite billions of years of evolution in these conditions, we typically study bacteria in mono-species cultures, at high concentrations and in nutrient rich conditions thus ignoring both inter-species interactions and phenotypic heterogeneity within bacterial populations. There is strong evidence to suggest that we are overlooking important aspects of microbial biology as a consequence, yet technologies to study bacterial populations in controlled, reproducible, high throughput conditions are significantly lacking. This thesis aimed to explore physical methods to control the development of microbial populations and to design microbial communities. Looking to recent developments in microfabrication and colloidal science techniques, we developed bio-sCAPA, a technique for patterning entire populations of bacteria on a surface with single-cell precision. In a first study, we apply bio-sCAPA to investigate phenotypic heterogeneity in an antibiotic tolerant population of Staphylococcus aureus and show that S. aureus tolerance to flucloxacillin and rifampicin killing is characterised by a delayed lag-time phenotype but not a heterogeneous growth-rate phenotype. In a second study, we explore Bacillus subtilis spores as a means of designing living soft materials with spores as the colloidal building block. We explore how we can control spore aggregation dynamics both in bulk and at oil-water interfaces and show that spores can be used to stabilise oil-water emulsions. We then show how bio-sCAPA can be used to design and control growing active nematic structures using filamenting B. subtilis cells. Turning towards species-species interaction and microbial communities, we study pair-wise interactions between three species living in the human upper respiratory tract with a view towards re-engineering the nose microbiome to prevent colonisation by Staphyloccocus aureus. We characterise the antagonistic mechanism between two commensal organisms, Dolosigranulum pigrum and Corynebacterium pseudodiphtheriticum, against S. aureus. We present evidence, using both macroscopic and microscopic techniques, that D. pigrum directly inhibits S. aureus via an as-yet unknown inhibition mechanism and we characterise this inhibition agent in terms of its killing effect and how easily S. aureus develops resistance to the inhibition agent. We further show that C. pseudodiphtheriticum both inhibits S. aureus in a nutrient-dependent manner and also augments D. pigrum growth, which directly leads to earlier onset of S. aureus inhibition by D. pigrum. Our study of a minimal microbial community thus demonstrates some of the complex, interdependent interactions taking place within the upper respiratory tract. In the final chapter, we turn to addressing certain key limitations of bio-sCAPA that centres primarily on desiccation susceptibility in certain microbes. We explore an alternative approach of patterning particles functionalised with nanobodies that selectively bind species of interest and show that it resolves the key desiccation bottleneck of bio-sCAPA. Further improvements to the strategies developed in this thesis are discussed in the conclusion and a section on potential future directions is presented.