This is, to the best of our knowledge, the first study to investigate plasmidomes at a global scale using long read sequencing from complex untreated domestic sewage. Previous metagenomic surveys have detected AMR genes in a variety of environments, including sewage.

Antimicrobial resistance is a global threat to human health, as it inhibits our ability to treat infectious diseases. This study utilizes sewage water plasmidomes to identify plasmid-derived features and highlights antimicrobial resistance genes, particularly macrolide resistance genes, as abundant in sewage water plasmidomes in Firmicutes and Acinetobacter hosts.

Sewage water from around the world contains an abundance of short plasmids, a number of which harbor antimicrobial resistance genes (ARGs). The global dynamics of plasmid-derived antimicrobial resistance and functions is only starting to be unveiled. Here, we utilized a previously created dataset of 159,332 assumed small plasmids from 24 different globally collected sewage samples. We investigated the detailed phylogeny as well as the interplay between their protein domains, ARGs, and predicted bacterial host genera to help understand the global sewage plasmidome dynamics. A total of 58,429 circular elements carried genes encoding for plasmid-related features, and MASH distance analyses showed a very high degree of diversity. A single very diverse cluster of 520 predicted Acinetobacter plasmids was predominant among the European sewage water. Based on functional domain network analysis, we identified three groups of plasmids, mainly replication and mobilization domains. However, these backbone domains were not exclusive to any given group. Acinetobacter was the dominant host genus among theta-replicating plasmids at these size ranges. They contained a reservoir of the macrolide resistance gene pair msr(E) and mph(E). Macrolide resistance genes were the most common resistance genes in sewage plasmidomes and found in the largest number of unique plasmids. While msr(E) and mph(E) were limited to Acinetobacter, erm(B) was disseminated among a range of Firmicutes plasmids, including Staphylococcus and Streptococcus, highlighting a potential reservoir of antibiotics resistance for these pathogens from around the globe.

Plasmids can provide a selective advantage for microorganisms to survive and adapt to new environmental conditions. Plasmid-encoded traits, such as antimicrobial resistance (AMR) or virulence, impact on the ecology and evolution of bacteria and can significantly influence the burden of infectious diseases. Insight about the identity and functions encoded on plasmids on the global scale are largely lacking. Here we investigate the plasmidome of 24 samples (22 countries, 5 continents) from the global sewage surveillance project. We obtained 105 Gbp Oxford Nanopore and 167 Gbp Illumina DNA sequences from plasmid DNA preparations and assembled 165,302 contigs (159,322 circular). Of these, 58,429 encoded for genes with plasmid-related and 11,222 with virus/phage-related proteins. About 90% of the circular DNA elements did not have any similarity to known plasmids. Those that exhibited similarity, had similarity to plasmids whose hosts were previously detected in these sewage samples (e.g. Acinetobacter, Escherichia, Moraxella, Enterobacter, Bacteroides, and Klebsiella). Some AMR classes were detected at a higher abundance in plasmidomes (e.g. macrolide-lincosamide-streptogramin B, macrolide, and quinolone), as compared to the respective complex sewage samples. In addition to AMR genes, a range of functions were encoded on the candidate plasmids, including plasmid replication and maintenance, mobilization, and conjugation. In summary, we describe a laboratory and bioinformatics workflow for the recovery of plasmids and other potential extrachromosomal DNA elements from complex microbiomes. Moreover, the obtained data could provide further valuable insight into the ecology and evolution of microbiomes, knowledge about AMR transmission, and the discovery of novel functions.