Title: Functional microbiomics
Description: Antibiotic resistance in human pathogens severely limits our ability to treat bacterial infections resulting in significant human suffering globally. A majority of antibiotic resistance genes have been acquired by pathogens through horizontal gene transfer. The origin of these genes is poorly understood, but reservoirs of resistance genes accessible to pathogens must exist. This dissertation investigates antibiotic resistance reservoirs in the environment and human microbiomes using culture dependent and independent techniques. Additionally the developed methods are applied to improve the tolerance of biofuel relevant bacteria towards important biomass inhibitors. First we show that bacteria capable of subsisting on antibiotics are widely distributed in the environment. These bacteria are phylogenetically closely related to pathogens and are highly resistant to most antibiotics, thus they constitute an unappreciated reservoir of antibiotic resistance that could be accessed by pathogens. We then show that bacterial isolates from the human microbiome are highly resistant to antibiotics and investigate the dynamics and inter-individual variation in microbiomic resistance profiles. We demonstrate that antibiotic resistance determinants can be exchanged between microbiome isolates and other strains illustrating that the human microbiome is a breeding ground and exchange of antibiotic resistance genes. To elucidate this antibiotic resistance reservoir we apply metagenomic functional selections to identify resistance genes from both cultured microbiome isolates and directly from fecal and sputum samples.

Strikingly the genes identified using culture dependent methods are highly similar to genes harbored by human pathogens whereas genes identified using culture independent methods are distantly related to resistance genes of pathogens. This highlights a reservoir of previously unknown resistance genes harbored by the uncultured fraction of the human microbiome and demonstrates that exchange of resistance genes between microbiome isolates and pathogens has occurred. We also identify 170 antibiotic resistance genes from bacteria capable of subsisting on antibiotics and show that 10% of these are identical at the nucleotide level to resistance genes harbored by pathogens demonstrating recent evolutionary exchange of resistance genes between these bacteria. Finally we apply metagenomic functional selections to identify novel genetic parts from the soil microbiome, that confer tolerance to important biomass inhibitors currently limiting sustainable biofuel production, providing proof of concept of how to make available Nature's diverse inventory to synthetic biology.