Mobile antibiotic resistome in wastewater treatment plants revealed by Nanopore metagenomic sequencing

In this article, Che et al. performed metagenomic sequencing of wastewater treatment samples to identify the hosts of antibacterial resistance genes and monitor their spread. Nanopore long-reads greatly facilitated the characterisation of multi-drug resistant plasmids, which furthered our understanding into the role of these plasmids in facilitating the survival of antibacterial resistant bacteria in wastewater treatment plants.

Background
Wastewater 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.

Results
In 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. Lastly, 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.

Conclusion
Overall, these findings substantially expand our current knowledge of the resistome in WWTPs, and help establish a baseline analysis framework to study ARGs in the environment.