Abstract
Antimicrobial resistance in bacteria poses a substantial threat to global health. The acquisition of antibiotic resistance genes (ARG) by bacteria is often mediated by plasmids, mobile genetic elements that can transfer between phylogenetically distant bacteria through cell-cell contact. One habitat where this gene exchange occurs is in farm or feedlot settings, as animal feces is known to contain a high number of bacteria with mobile ARG. Via the application of manure on agricultural soil, mobile ARG can spread further to bacteria present in soil, water and crops. While manure and agricultural soil have been recognized as sources of antibiotic-resistant bacteria and their ARG, important knowledge gaps remain regarding the soil bacteria that acquire resistance plasmids from manure. Identification of these bacteria is pivotal, as they can become reservoirs of ARG and subsequently facilitate their spread to human pathogens. This thesis aims to address existing knowledge gaps by developing and applying novel metagenomic tools to better monitor the presence and spread of plasmids in manure and agricultural soil. In chapter one, we outline the global significance of antimicrobial resistance with a focus on plasmids as key players in the dissemination of ARG. We explore the interconnectedness of habitats and discuss factors that influence the ecology and evolution of plasmids. In chapter two, we develop a novel method, Hi-C+, to identify rare plasmid hosts in a soil microbial community. This method enables the identification of plasmid hosts by creating physical linkages between plasmid and chromosomal DNA within a bacterial cell. This association is lost by traditional metagenomic approaches yet is crucial for understanding the ecology of plasmids in microbiomes. In chapter three, we implement Hi-C+ to monitor the transfer of a multi-drug resistance plasmid from manure to the rhizosphere of barley, the soil adjacent to the plant roots. We detect plasmid transfer and identify rare soil microbiome members as potential new plasmid hosts, suggesting that hitherto uncharacterized, and possibly unculturable bacteria could play an important role in the dissemination of multi-drug resistance plasmids from manure to agricultural soil. Lastly, the fourth chapter details the use of long-read sequencing to identify the replicons on which ARG reside in manure. ARG carried on plasmids pose a higher threat for mobilization to human pathogens compared to chromosomally encoded resistance. We demonstrate a lack of congruence between publicly available tools used to detect plasmids in metagenomes and identify the tool that achieves the best performance. We also show that ARG are prevalent on plasmid and chromosomal sequences and that there is a clear effect of enrichment culture bias in profiling the ARG diversity in manure. This study underscores the power of long-read sequencing in co-localizing multiple ARG on the same replicon and in distinguishing plasmid versus chromosomal backgrounds. Altogether, this thesis contributes to a multifaceted understanding of plasmid-mediated antimicrobial resistance, with a focus on plasmid spread in agricultural settings.