Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

Leon Peto,Martin J. Llewelyn,Derrick W. Crook,Tim E. A. Peto,Nicola J. Fawcett,A. Sarah Walker,Yung-Fu Chang

doi:10.1371/journal.pone.0222831

Abstract

Metagenomic sequencing of fecal DNA can usefully characterise an individual's 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 feces 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 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 fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

Highlights

The intestinal microbiome is one of the most important reservoirs of clinically relevant antimicrobial resistance (AMR) genes [1]
Use of the QuickLyse plasmid extraction method increased the number of reads mapping to plasmid-associated beta-lactamases to a mean of 15.7 Reads per Kb per million (RPKM) compared to a mean of 4.4 RPKM using the Fast DNA Stool kit (S1 Fig)
Six hours was selected as the optimal incubation period, as this provided approximately 104-fold enrichment while preserving the ratios of the different E. coli strains present at baseline

Summary

Introduction

The intestinal microbiome is one of the most important reservoirs of clinically relevant antimicrobial resistance (AMR) genes [1]. Quantitative culture requires further genotyping to define resistance elements and can only practically be used for a small number of species present in feces [6], and targeted molecular approaches, such as qPCR, cannot simultaneously detect the dozens of AMR genes and mutations that may be present in a sample from among thousands of known types [7,8] Because of these shortcomings, metagenomic sequencing performed directly from extracted fecal DNA is increasingly being used to characterise the gut resistome, as it produces millions of short DNA reads that can be matched to a catalogue of thousands of AMR genes, theoretically producing a representative, quantitative description of AMR genes in a sample [1,7,9]

Objectives

Methods

Results

Discussion

Conclusion