Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing

Abstract

Background: Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for < 1% of all sequenced DNA, leading to limitations in detection of low-abundance resistome-virulome elements. This study describes the extent and composition of the low-abundance portion of the resistome-virulome, using a bait-capture and enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias.

Results: The use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins.

Conclusions: These results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

Keywords: Antimicrobial resistance; Microbial ecology; Molecular enrichment; Rare microbiome; Resistome.

Fig. 1

Venn diagram depicting the number of gene accessions (a) and gene groups (b) identified in enriched libraries only (green, N = 32 libraries); non-enriched libraries only (purple, N = 32 libraries); or both enriched and non-enriched libraries (overlapping blue areas, N = 64 libraries)

Fig. 2

One hundred percent stacked graphs of resistome/virulome composition, by sample type and by library preparation assay (R-UMI Resistome-UMI, R Resistome, M-UMI Metagenome-UMI, M Metagenome, E composition in portion of the resistome identified only through enrichment). Proportional abundances were calculated by dividing the number of de-duplicated hits to each class by the total number of de-duplicated hits. Classes are shown individually if they contained at least 10% relative abundance in one of the assays within each sample type; all other classes were grouped into the “Other” category

Fig. 3

Binary heatmap of betalactamase groups identified only in enriched sequence libraries (N = 32). Gray present, white absent

Fig. 4

a–d Nucleotide variant depiction for the ermG gene (the length of which is represented on the x-axis) across all 64 samples (rows), grouped by library preparation assay (a–d) and sample type. Blue lines indicate SNPs identified in all 4 samples within sample type and assay, while red lines highlight SNPs that uniquely identify the sample type in which they are found by assay

Fig. 5

NMDS ordination of resistome composition at the class level, for a the entire resistome (i.e., all gene accessions identified) and b the low-abundance resistome (i.e., the additional gene accessions identified via enrichment). Each mark is one sample, mark color indicates sample type (blue beef, orange poultry, red swine, gray WWTP), and mark shape indicates library prep assay (circle Resistome-UMI, diamond Resistome, asterisk Metagenome-UMI, triangle, Metagenome). In both a and b, ordination by sample type was significant with a large effect size (R = 0.73 and P < 0.001 for both), while ordination by library preparation assay was significant but with a small effect size in a (R = 0.13 and P < 0.001) and insignificant in b (R = 0.54, P = 0.12). Three dimensions were used for ordination

Fig. 6

Boxplots of proportion of sequencing reads on-target (log-scale), by assay. Each dot is a sample (n = 64), colored by type (blue beef, orange poultry, red swine, gray WWTP). Boxes represent interquartile range and median, and whiskers represent range, except for outliers. Assays with different letters were significantly different (Tukey P < 0.05)