Mobile antibiotic resistome in WWTPs and the mechanisms for horizontal transfer of resistance genes

Abstract

This thesis is centered around the problem of environmental dimension of antibiotic resistance. I employed an interdisciplinary approach of next-generation sequencing, bioinformatics and molecular techniques to understand the interactions between different mobile genetic elements (MGEs) and the major contributors in facilitating the transfer of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs).

I developed a pipeline based on Nanopore metagenomic sequencing to comprehensively reveal the genetic context and track the hosts of ARGs along the wastewater treatment process. I found that most of the ARGs were carried by plasmids and interestingly, integrative and conjugative elements (ICEs) carrying five types of ARGs were detected and a broad spectrum of ARGs carried by plasmids (29 subtypes) and ICEs (4 subtypes) were persistent across the WWTPs. Host tracking showed a variety of potential antibiotic-resistant bacteria in the effluent. Given the close association between MGEs and resistance, I systematically investigated the interaction of different MGEs in mediating the transfer of ARGs by in silico genomic analysis of a large collection of complete plasmids (14,029) and genomes (12,059), and then confirmed the interactions using direct experimental approaches. I developed a tool (Plas-CAD) for plasmid classification, ARG annotation and visualization, and found that most plasmid-borne ARGs, including those localized on class 1 integrons, were enriched in conjugative plasmids. Notably, I reported the discovery and characterization of a massive insertion sequence (IS)-associated ARG transfer network (245 combinations covering 59 ARG subtypes and 53 ISs) linking conjugative plasmids and phylogenetically distant pathogens, suggesting a general mechanism for the horizontal transfer of ARGs mediated by the interaction between conjugative plasmids and ISs.

To investigate the prevalence of ARG-bearing conjugative plasmids in WWTPs, I constructed an E. coli K12 MC1061-Rif+::mClover3-Km+ recipient to capture conjugative plasmids conferring resistance to a variety of antibiotics from WWTPs. Hybrid assembly (Nanopore and Illumina) of a total of 31 captured conjugative plasmids revealed a high rate of co-occurrence of different ARGs. Importantly, some ARG-bearing plasmids were highly similar to those isolated from animal/human sources, highlighting the significance of conjugative plasmids in mediating the co-transfer of large repertoires of ARGs.

Finally, I applied population genomics to confirm the contribution of plasmids to the development of multidrug-resistance (MDR) and seek potential epidemiological links of resistance between WWTPs and human/animal associated bacteria. I found that most WWTP isolates were genetically distinct from their closest known relatives of human/animal associated strains. Even in the minority of lineages that were closely related, WWTP isolates were characterized by quite different plasmid compositions. Moreover, I identified a high diversity of complete plasmids (n = 264, 141 were novel), which served as the main source of resistance, and showed ARG-bearing plasmid transfer across ecological boundaries. Importantly, the close association of plasmid-borne ARGs with ISs was also observed in these MDR isolates. These findings suggested that transfer of MDR between WWTPs and human/animal bacteria was linked through a multi-layered hierarchical process, in which the synergy between plasmids and ISs played an important role and the ISs-associated ARGs served as a basic transfer unit.