Wastewater treatment plants (WWTPs) have been recognised as reservoirs for antibiotic resistance and environments in which human pathogens commonly dwell all the time given that WWTPs constantly collect residual pharmaceutical compounds and massive amounts of human-gut associated bacteria. As recurrent antibiotic selective pressure and high cell densities favour horizontal gene transfer for dissemination of antibiotic resistance genes (ARGs) and considering the possibility of ARGs transmission from wastewater microbial communities to human pathogens, it is highly important to reveal and assess the potential barrier to prevent potential human health risks. However, the bulk of our current knowledge on the prevalence and abundance of the main driver of the horizontal gene transfer – mobile genetic elements (MGEs) is limited to those in databases and previous studies. Therefore, we have little overall information about the extent of participation and niche preference of the MGEs in a complex environment such as the wastewater ecosystem.
In this PhD thesis, we targeted three urban water systems located in Denmark, Spain, and the United Kingdom to decipher the dynamics and fate of the total pools of MGEs, namely the mobilome. This thesis is an attempt to ascertain the general features and distinct patterns of the wastewater mobilome and its associated antibiotic resistome with the dynamic changes of wastewater treatment stages – from the sewer sources to the interior biological treatment processes in the WWTPs. Three European countries with different antibiotic use practices were considered, and sampling campaigns were constructed in two seasons (summer and winter, 2018). 16S-rRNA amplicons, high-throughput qPCR array, exogenous plasmid isolation, permissiveness test, mobilome and metagenome were produced from the recruited sewer and wastewater samples for the different types of data to shed light on the interaction of microbiome, mobilome and antibiotic resistome. Furthermore, metadata containing physicochemical, hydrology and other parameters of the related sampling campaigns was utilized for linking and exploring different aspects of the dataset. The thesis 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. This thesis has resulted in three manuscripts, along with several collaborations that have not been listed here, which hopefully will lead to further publications and new investigations.
Preliminarily, in a collaborative study using the same constructed samples “Extended-spectrum beta-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage” (DOI: 10.1021/acs.est.0c08548), we have systematically quantified 70 extended-spectrum beta-lactamase (ESBL) and carbapenemase genes with spatiotemporal variations. This study gives us the message that the urban water systems harbour persistent clinically important ARGs, and they were correlated with the fate of distinct taxonomic groups and MGEs. In order to understand the interplay of ARGs and MGEs dynamics across the treatment compartments, we primarily paid attention to the ARGs associated MGEs, particularly plasmids. That is because plasmids are able to shape bacterial evolution as key HGT molecular vehicles that have efficient and rapid transmission capacity, and plasmids can be involved in various HGT mechanisms such as conjugation, transduction and transformation. In Manuscript 1 “Horizontal transmission of a multidrug-resistant IncN plasmid isolated from urban wastewater”, we used a fluorescent-reporter-gene based exogenous isolation approach to capture ESBL encoding mobile determinants from sewer microbiome samples that enter an urban water system in Denmark. After screening and sequencing, we isolated one complete 73,132 Kbp circular contig and it was subsequently characterized as an IncN plasmid carrying a complete set of functional conjugative genes. Furthermore, this plasmid showed multiple antibiotic resistance with corresponding ARGs. This plasmid was then gfp tagged and tested for permissiveness retransferring to the wastewater communities. As a result, the potential host range of this plasmid was revealed. In addition, a sequence similarity search across curated plasmid repositories revealed that this IncN plasmid derives from an IncN backbone harboured by environmental and nosocomial Enterobacterial isolates. Finally, the abundance of this IncN plasmid was measured using a short-reads mapping from shotgun metagenomic datasets from four locations in three European countries. This plasmid displayed the highest relative abundance in the hospital sewers than in any other compartments across all countries.
Based on the findings of the occurrence and variance of an IncN plasmid encoding multiple ARGs in the urban water systems, we were curious about the fate of other plasmids and moreover, the plasmidome (total pool of plasmids) and its derived ARGs along with the treatment processing. In Manuscript 2 “Insights into the circular: the cryptic plasmidome and its derived antibiotic resistome in the urban water systems”, we employed a direct plasmid DNA metagenome sequencing strategy skipping the widely used transposon-aided capture and multiple displacement amplification and aiming to uncover larger-sized plasmids. We processed the raw data using our Plaspline pipeline (version: v1.1) in the circular module to generate ascertained circular-topology plasmid contigs. Through this approach, we recovered 10,942 non-redundant putative plasmid contigs representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups, of which 87% were novel plasmids compared to the PLSDB database. Plasmid size-diversity distribution benchmarked the feasibility and reliability of the methodology. We presented that the highest prevalent plasmids were the non-mobilisable ones and the plasmidome richness generally decreased with the treatment processing. Plasmid communities could be clearly clustered based on the treatment compartments, while the seasonal variations seemed not influential. Plasmids encoding ARGs were dominantly mobilisable, and the predominant plasmid-borne ARGs targeted resistance to aminoglycosides (ant, aph), beta-lactams (blaOXA, blaSCO and blaTEM) and tetracycline (tet). To our surprise, there was a common plasmid pool shared by the three countries, and a few plasmids were persistent across the different treatment stages. Finally, we showed some persistent plasmids carried ARGs, implying persistent plasmid-mediated antibiotic resistance emerged in the pan-European urban water systems.
Since the focus of Manuscript 2 was on circular-topology plasmidome and consequently, the derived resistome analysis was made exclusively on the circular plasmid contigs, the image of those small linear plasmid contigs was lacking. Regarding Manuscript 3 “Plasmidome derived antibiotic resistome reveals the partitioning of different geographic regions and treatment compartments in the urban water systems”, we applied the same sequencing output from Manuscript 2 considering that our rigorous experimental methodology has recovered almost pure plasmid DNA from the sewer/wastewater samples. Accordingly, we considered the assembled circular and linear contigs from this pool were all plasmids (at least predominantly, there may be other MGEs included), while the linear contigs were not circularized due to sequencing depth or intrinsic unique genetic contexts. We removed the chromosome noise and accordingly, generated the target plasmidome in this study. We identified 225 ARGs belonging to 180 groups of ARG families in this ‘full version’ plasmidome. The plasmid resistome richness and relative abundance detected in Spanish UWS samples were significantly (p < 0.05) higher than the other studied countries, which mirrors the remarkable domestic antibiotic use in Spain. And we only detected significant (p < 0.05) differences in ARG risk scores between the hospital and residential sewers in Spain. Meanwhile, different sewer compartments showed a partitioning role for the resistome richness and abundance distributions. Intriguingly, we perceived a group of shared ARGs among the three countries regardless of treatment stages. Further, ≥ 80% of ARG types in the wastewater treatment plants could be found in the sewer sources, implying these ARGs were persistent in the urban water systems. Correlations analyses were performed for linking the plasmidome derived resistome, microbiome, and the total resistome (documented by a high-throughput qPCR array). Overall, Manuscript 3 demonstrates the important role of plasmidome derived resistome in the total antibiotic resistome, and such plasmidome resistome was shaped by geographic-regional and treatment-sectional variations in the urban water systems.
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