Selective culture enrichment and sequencing of faeces to enhance detection of antimicrobial resistance genes

Abstract

Metagenomic sequencing of faecal DNA can usefully characterise an individuals intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer faeces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction protocols increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a faecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.