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.
Bibliographical noteFunding Information:
This research was supported by the USDA NIFA awards 2015-68003-23048 (awarded to Paul S. Morley, Keith E. Belk, Kenneth L. Jones, Christina A. Boucher, Jaime Ruiz, and Noelle R. Noyes) and 2016-67012-24679 (awarded to Noelle R. Noyes, Zaid Abdo, Christina A. Boucher, Paul S. Morley, and Keith E. Belk). The funders had no role in design of the study or in collection, analysis and interpretation of data, or in writing the manuscript.
- Antimicrobial resistance
- Microbial ecology
- Molecular enrichment
- Rare microbiome