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


Autoři: Leon Peto aff001;  Nicola J. Fawcett aff001;  Derrick W. Crook aff001;  Tim E. A. Peto aff001;  Martin J. Llewelyn aff004;  A. Sarah Walker aff001
Působiště autorů: National Institute for Health Research (NIHR) Health Protection Research Unit on Healthcare Associated Infections and Antimicrobial Resistance, John Radcliffe Hospital, Oxford, England, United Kingdom aff001;  Nuffield Department of Medicine, University of Oxford, Oxford, England, United Kingdom aff002;  National Infection Service, Public Health England, Colindale, London, England, United Kingdom aff003;  Department of Global Health and Infection, Brighton and Sussex Medical School, Falmer, Sussex, England, United Kingdom aff004;  Department of Microbiology and Infection, Brighton and Sussex University Hospitals NHS Trust, Brighton, England, United Kingdom aff005
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222831

Souhrn

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.

Klíčová slova:

Antimicrobial resistance – DNA extraction – DNA sequencing – Enterobacteriaceae – Gene sequencing – Staphylococcus aureus – Vancomycin


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Článek vyšel v časopise

PLOS One


2019 Číslo 11