Discovery And Evolution of Metagenomic Antibiotic Resistance Genes

The observed overlap between antibiotic resistance genes found in soil microbes and those emerging in clinical settings has spurred researchers to functionally screen the metagenomes of soil bacteria to: (1) gain insight into the prevalence of resistance genes in our environment, and (2) identify potential new antibiotic resistance mechanisms. The information obtained from these studies is useful for identifying resistance mechanisms likely to arise in the clinic and informing stewardship measures to counter them. However, traditional methods for functionally screening metagenomic DNA have two main limitations. Firstly, any model bacterial host can only effectively transcribe and translate a minority of environmental genes. In nature, potential resistance genes may be transferred by mobile elements that enable high-level expression, but replicating this in laboratory settings is challenging. Our lab team has addressed this issue by developing a cloning method that promotes high-level expression of captured genes in E. coli, allowing for increased detection of metagenome-acquired phenotypes compared to standard metagenomic libraries. The second notable limitation of traditional functional screening is that it only detects mature resistance genes, leaving “primordial” resistance genes undetected. When exposed to antibiotics, these weaker resistance genes may require only a few mutations to confer high-level resistance, and thereby still pose a substantial clinical threat. The research in this thesis describes the development of a functional screening pipeline using a metagenomic library created with the aforementioned cloning strategy to uncover primordial resistance elements, and resistance elements not previously observed in clinical pathogens. I showed that this pipeline could incorporate a mass double-plating step that provides an efficient way to eliminate resistance due to spontaneous mutation rather than metagenome-acquired genes. This led to the recovery of 15 chloramphenicol resistance elements, 11 tigecycline resistance elements, and 11 meropenem resistance elements. Additionally, directed evolution was employed to assess the evolvability and potential threat of two metagenomic resistance elements: a phosphotransferase that confers chloramphenicol resistance and a vicinal oxygen chelate protein that confers tigecycline resistance. Importantly, the phosphotransferase didn’t reach the level of resistance of current clinically relevant resistance proteins, suggesting it is not an immediate clinical threat. In contrast, the vicinal oxygen chelate protein was able to confer tigecycline resistance beyond the FDA breakpoint upon on one substitution. Lastly, collateral resistance testing revealed that the phosphotransferase also confers resistance to thiamphenicol, but not florfenicol, meaning that if the phosphotransferase were to become a clinical threat, florfenicol would likely be a suitable alternative to chloramphenicol. Collateral resistance testing also revealed that if the vicinal oxygen chelate protein were to become a clinical tigecycline threat, eravacycline or omadacycline would be suitable alternatives.