Linking the resistome and plasmidome to the microbiome

Abstract

The rapid spread of antibiotic resistance among bacterial pathogens is a serious human health threat. While a range of environments have been identified as reservoirs of antibiotic resistance genes (ARGs), we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the natural bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrate that the in vivo proximity-ligation method Hi-C can reconstruct a known plasmid-host association from a wastewater community, and identify the in situ host range of ARGs, plasmids, and integrons by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile genetic elements and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the broadest host range in this wastewater, and identified bacteria belonging to Moraxellaceae, Bacteroides, and Prevotella, and especially Aeromonadaceae as the most likely reservoirs of ARGs in this community. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.

Fig. 1

Hi-C deconvolution workflow. a Formaldehyde induces covalent bonds between DNAs that are close in three-dimensional space (bonds are depicted by black arcs), and therefore within the same cell. b Hi-C library preparation via restriction enzyme digestion (top left), ligation and junction enrichment (bottom left), preparation of the fragment for paired-end sequencing (top right), reads from fragments generated by Hi-C provide linkage information of non-contiguous DNA originated from the same cell (bottom right). c Hi-C links can be used in a graph clustering context to deconvolute contigs into their original cellular groupings, including both chromosomes and plasmids

Fig. 2

Hi-C links between the clusters and plasmid markers, integrons, and ARGs, identified in the WW sample and affiliated with Alpha- Beta-, Gamma-, and Delta-Proteobacteria. Each tip of the phylogenetic tree represents a cluster. For clarity only clusters having a contact with plasmid markers, integrons, or ARGs are shown (results showing all clusters are presented in Figs. S6 and S7). The presence or absence of a link is shown on the heatmap to the right of the tree, and the color shading represents the intensity of the normalized Hi-C link signals. a Aeromonadaceae were identified as a natural reservoir of ARGs. b Clusters affiliated with the genus Acinetobacter showed high Hi-C linkage to ARGs conferring resistance to aminoglycosides, beta-lactams, tetracycline, phenicol, and macrolides. c Most plasmids detected belonged to clusters related to Enterobacteriaceae. d As expected, BHR plasmids were linked to clusters with phylogenetic affiliations broader than the NHR plasmids. e Class 2 and 3 integrons were associated with clusters affiliated with the Neisseriaceae. The arrow indicates a cluster in Comamonadaceae which had a strong link to an IncP-1β plasmid

Fig. 3

Hi-C linkage between plasmid markers, integrons, and ARGs among clusters belonging to Bacteroidetes in the wastewater sample WW. Each tip of the phylogenetic tree represents a cluster. The presence or absence of a link is shown in the heatmap, with the shading representing the intensity of the normalized Hi-C linkage signal

Fig. 4

Hi-C linkage between plasmid markers, integrons, and ARGs among clusters belonging to Firmicutes in the wastewater sample WW. Each tip of the phylogenetic tree represents a cluster. The presence or absence of a link is shown in the heatmap, with the shading representing the intensity of the normalized Hi-C linkage signal

Fig. 5

Taxonomic assignment of contigs that were linked to contigs harboring plasmids, ARGs, or integrons. Here the strength of Hi-C linkage is represented by the length of the bars and is summarized by Phylum (pink, Actinobacteria; blue, Bacteroidetes; orange, Firmicutes; green, Proteobacteria, and gray: others), by Class (color shading), and stacked bars of the same color represent different families. *: ARGs or plasmids which did not have links to cluster when using our first approach. The cross indicates that the gene, or marker, was found in the same class when using our first approach