Abstract
BackgroundWastewater treatment plants (WWTPs) are recognized as hotspots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Despite our understanding of the composition and distribution of ARGs in WWTPs, the genetic location, host, and fate of ARGs remain largely unknown.ResultsIn this study, we combined Oxford Nanopore and Illumina metagenomics sequencing to comprehensively uncover the resistome context of influent, activated sludge, and effluent of three WWTPs and simultaneously track the hosts of the ARGs. The results showed that most of the ARGs detected in all compartments of the WWTPs were carried by plasmids. Transposons and integrons also showed higher prevalence on plasmids than on the ARG-carrying chromosome. Notably, integrative and conjugative elements (ICEs) carrying five types of ARGs were detected, and they may play an important role in facilitating the transfer of ARGs, particularly for tetracycline and macrolide-lincosamide-streptogramin (MLS). A broad spectrum of ARGs carried by plasmids (29 subtypes) and ICEs (4 subtypes) was persistent across the WWTPs. Host tracking showed a variety of antibiotic-resistant bacteria in the effluent, suggesting the high potential for their dissemination into receiving environments. Importantly, phenotype-genotype analysis confirmed the significant role of conjugative plasmids in facilitating the survival and persistence of multidrug-resistant bacteria in the WWTPs. At last, the consistency in the quantitative results for major ARGs types revealed by Nanopore and Illumina sequencing platforms demonstrated the feasibility of Nanopore sequencing for resistome quantification.ConclusionOverall, these findings substantially expand our current knowledge of resistome in WWTPs, and help establish a baseline analysis framework to study ARGs in the environment.
Highlights
Wastewater treatment plants (WWTPs) are recognized as hotspots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs)
Plasmids and integrative and conjugative elements (ICEs) carrying ARGs dominate the resistome in wastewater treatment plants (WWTPs) As shown in Additional file 1: Table S2, 1791 ARGs-carrying Nanopore reads with an average length of 8.5 kb were identified, much more than the 316 ARGs-carrying contigs assembled from Illumina short reads, indicating the difficulty to assess genetic context of ARGs by assembling Illumina short reads into long contigs
Our results demonstrated that a high diversity of ARGs persisted through the treatment processes in WWTPs, and their association with plasmids and ICEs might have a large contribution to the spread of ARGs
Summary
Wastewater treatment plants (WWTPs) are recognized as hotspots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Wastewater treatment plants (WWTPs), as a unique interface between humans and environments, harbor a large microbial genetic diversity, facilitating the exchange of ARGs by horizontal gene transfer (HGT) [1]. EpicPCR was developed to link functional genes and phylogenetic markers, such as 16S rRNA in single cells, and has been used to identify the hosts of ARGs [11]. This PCR-based method requires the sequence information of the target genes of interest, and only one functional target gene can be sorted each time, the phylogenetic marker gene could be a universal one [17]. A robust method is urgently required to resolve both the genetic environments and the hosts of the ARGs in a high-throughput format
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