A Peek into the Plasmidome of Global Sewage

Abstract

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 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 NextSeq DNA sequences from plasmid DNA preparations and assembled 165,302 contigs (159,322 circular). Of these, 58,429 carried genes encoding for plasmid-related and 11,222 for 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) 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. IMPORTANCE 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. However, it is unknown whether the AMR genes were present on the microbial chromosome or located on extrachromosomal elements, such as plasmids. Using our approach, we recovered a large number of plasmids, of which most appear novel. We identified distinct AMR genes that were preferentially located on plasmids, potentially contributing to their transmissibility. Overall, plasmids are of great importance for the biology of microorganisms in their natural environments (free-living and host-associated), as well as for molecular biology and biotechnology. Plasmidome collections may therefore be valuable resources for the discovery of fundamental biological mechanisms and novel functions useful in a variety of contexts.

Keywords: Illumina NextSeq; Oxford Nanopore; Oxford Nanopore sequencing; animals; human; microbiome; plasmid DNA isolation; plasmid reconstruction; plasmids; wastewater.

FIG 1

Schematic overview of the single read assembly workflow and size distribution of assembled reads. (A) Nanopore reads (based on plasmid DNA amplified with phi29) longer than 10,000 bases were split into 1,500-base fragments. The sequence fragments were then assembled using minimap2 and miniasm and subsequently polished two times: with the Nanopore fragments using racon and with the Illumina reads using pilon. (B) Size distribution of circular (orange) and linear (violet) assembled elements. These are the candidate plasmid sequences that successfully mapped to the original Nanopore read (i.e., covering more than 60% of the read and not overlapping by more than 50 bp for multiple hits). Of the total 165,302 assemblies, 159,322 were characterized to be circular and 5,980 were characterized to be linear.

FIG 2

Functional characterization of circular DNA elements based on protein families. (A) Bar plot displaying the fraction of Pfam identifiers assigned to predicted proteins on the circular elements. The 31 Pfam identifiers represent the Top10 Pfam identifiers for each sample. Protein domains specifically involved in plasmid mobilization and plasmid replication are indicated in red and blue, respectively (see legend to the bottom right). Virus/phage-related Pfam identifiers are indicated in green. Remaining Pfam identifiers are grouped (other) and are indicated in dark gray. (B) Data for proteins with a Rep_3 (PF01051) domain (n = 24,824) were combined with the 1,637 reference Rep_3 (PF01051) proteins from Pfam. The protein sequences with a length of ≥40 amino acids (n = 16,930) were aligned using MAFFT. A phylogenetic tree was build using FastTree and visualized using FigTree. A high-resolution version of the phylogenetic tree is available from Figshare at https://doi.org/10.6084/m9.figshare.14112992.

FIG 3

Comparison of candidate plasmids from global sewage with known plasmids in the plasmid database (PLSDB). (A) Heat map of centered log ratio (clr)-transformed abundancies of plasmid candidates assigned to plasmids in the PLSDB at the bacterial genus level. The phylum level is indicated by letters in in parentheses: A, Actinobacteria; B, Bacteroidetes; aP, Alphaproteobacteria; bP, Betaproteobacteria; gP, Gammaproteobacteria; F, Firmicutes. Clustering of samples was performed using the Euclidean distances of the clr-transformed values. (B) Principal-component analysis of clr-transformed abundancies of known plasmids detected by the PLSDB. The plot at the top reveals similarities and differences between samples. The plot at the bottom reveals the known plasmids that drive the partitioning of the samples, with 17.6% of the variation explained by the first and 11.1% by the second principal component.

FIG 4

Antimicrobial resistance profiles from the whole community and plasmidomes from global sewage. (A) Bar plot displaying the proportions of antimicrobial resistance classes detected in a ResFinder-based analysis using the Illumina reads from the whole community, as well as Illumina reads from the plasmid preparations and Nanopore reads from the plasmid preparations. (B) Six examples of candidate plasmids are visualized in plasmid maps. The outermost black circle indicates the plasmid chromosome, and the coding sequence regions are colored according to their predicted function: replication (blue), mobilization (violet), transposition of DNA (green), antimicrobial resistance (red), toxin-antitoxin systems (orange), and hypothetical proteins (hp) and other proteins (gray). Blue and green lines indicate the GC and AT contents, respectively. The plasmids are named according to their origin: CIV (Côte d’Ivoire), POL (Poland), USA.1 (USA), BRA (Brazil), CZE (Czech Republic), and IND (India). Some sequencing errors might still be present in the candidate plasmid sequences, which are likely the reason why a few open reading frames are not properly predicted and appear fragmented, such as the gene encoding AmpC and macrolide efflux pump genes in the plasmid from Czechia. A detailed description about the plasmids is available from Figshare at https://doi.org/10.6084/m9.figshare.14039390.