Using Nanopore Sequencing Deciphering Antibiotic Resistance Dissemination In The Wastewater Systems

There is mounting concern that wastewater sewer systems, as the receptacle for excreted antibiotic resistance genes (ARGs), and horizontal gene transfer. Antimicrobial resistance genes readily transfer from pathogens and commensal hosts in human wastes to environmental strains and are better adapted to the sewer environment, which is efficiently driven by conjugative plasmid transfer. The WWTP influents often contain sewage of both domestic origin and hospital sources, which mainly harbour significant abundance and diversity of ARGs. Under the high pressure of antibiotic selection, the load of multi-resistant bacteria and genes in hospital sewage was higher than theirs in the domestic sewage, which potentially stimulate the transfer of ARGs from pathogens to environmental bacteria. It is however not clear to what extent the plasmid can act as a transmitter to facilitate the ARGs dissemination in this procedure. Conjugation, the most responsible HGT mechanism for mobilizing resistance among bacteria, might cause further dissemination of ARGs. Conjugative plasmids, encoded with multiple resistance genes, allow the acquisition of multidrug resistance in transfer and make an antibiotic selection from one to all. Understanding the fate of ARGs encoded on conjugative plasmids in sewage is therefore essential to disentangle its roles in ARG dissemination. Expect the surveillance of sewage inlets, the receiving water body from WWTP has also aroused concerns related to human health problems. Wastewater treatment plant (WWTP) effluent discharge could induce resistome enrichment in the receiving water environments. The downstream receiving water body of the WWTP was observed as an important reservoir for the transmission of pathogenic bacterial hosts with alarming resistance levels. Understanding the driving force shaping such resistome booming in receiving environments is essential to establish priority in mitigating ARG spread and evaluating health risks. However, this question cannot be fully elucidated without quantitating source tracking based on precise and extensive identification of the host populations of ARGs.

 This thesis was mainly to apply the pioneering sequencing technology-Oxford Nanopore Technologies to solve the problems mentioned above, which is principally divided into two chapters. The Introductory chapter explains the detailed background, aims, and hypothesis of the research targets. And in the Manuscript chapter, the main subjects, results, and opinions are thoroughly covered. The works in Manuscript I seek to decipher the fate of conjugative plasmids facilitating the dissemination of ARGs in the sewer influent to WWTPs. The sewer influent samples were collected from hospital sewers and residential sewers located in Denmark, Spain, and the United Kingdom. Three European countries with different antibiotic use practices were considered, and sampling campaigns were constructed in the winter, of 2018. We attempt to ascertain the general features and distinct patterns of the conjugative plasmids from different sewer sources. The culture-based nanopore barcoding sequencing method was introduced into this study, including the steps of exogenous plasmid isolation, permissiveness test, antibiotic susceptibility test, and nanopore barcoding whole genomic sequencing. These isolated conjugative plasmids displayed higher transfer efficiency in the hospital sewers than in the residential sewers across all countries and showed multiple antibiotic resistance with corresponding ARGs. Furthermore, the co-occurrence network analysis via Fisher exact test profiled the relationship among incompatibility genes, mobility groups, and ARGs encoded on the conjugative plasmids. We characterized the resistance phenotypes and genotypes carried by conjugative plasmids, especially those discharged from hospital and residential settings during conveyance in the sewers. Such a worrisome situation needs to be considered during sewage-based antibiotic resistance surveillance.

 The works in Manuscript II seek to understand the mechanism arousing the environmental resistome reservoir of coastal receiving water communities. This study audaciously applied nanopore sequencing technologies to comprehensively investigate the dissemination and enrichment of ARBs. A robust bioinformatic framework (ARGpore2) was constructed and carefully tested to predict resistance and establish plasmids/MGE-associated mobility. An overall consistent resistome was observed between Illumina-based and nanopore-based metagenomic approaches. However, compared to Illumina-assembled contigs, nanopore sequencing datasets generated a more reliable ARB profile that resistome of beta-lactamase carried by Escherichia overlooked by Illumina-assembled approach was accurately detected by nanopore sequencing, highlighting the power of assembly-free long-reads metagenomic approaches for assessing the mobile proportion of the resistome. The results suggested the receiving coastal system might be considered as a reservoir for ARGs mediated by the robust installation of WWTP-borne ARB community coupled with recombination and HGT enabled by plasmids and class 1 integrons. Consequently, in order to mitigate resistance pollution in receiving environments, advanced filtration or oxidative process should be included in the disinfection step of WWTPs to enable more complete disruption of pathogen cells and endospores during treatment.

 Manuscript III and Manuscript IV were mainly focused on exploring the application of nanopore technologies in deciphering the dissemination of ARGs in a special clinical environment (air dust in a hospital) and the functional genes encoded on extreme environmental (permafrost soil) microorganisms. In Manuscript III, we integrated a state-of-the-art metagenomic method to link ARGs to specific bacteria and we also confirmed the ARG transfer using cultural isolate whole genome Nanopore sequencing. Even though the whole study plan worked (from wet lab to data interpretation), we speculated that our survey (two-time points) only demonstrated limited ARG profiling and transfer information, meaning multiple survey years and more sampling frequencies (e.g. weekly or monthly) are needed. Additionally, Illumina sequencing still suffers from fragmented assembly and needs more turnaround time. The short-read sequencing methods were not able to address repetitive insertion sequences, which is often not avoidable in ARG regions. In Manuscript IV, to address the large data loss caused by the metagenomic assembly, we introduce FUNpore, a long-read correction and annotation framework. Such a correction-based annotation strategy showed promising precision and recall in functional annotation and could provide more permafrost microbial information. On-site MinION metagenomic sequencing of high-altitude permafrost soil showed that although 78.4% of genera were shared among vertical alpine, nitrate concentration, and solar radiation can lead to a certain level of community differentiation.

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