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

Autor: Nicola J Fawcett, Leon Peto, Tim E. A. Peto, Martin J. Llewelyn, A. Sarah Walker, Derrick W. Crook
Rok vydání: 2019
Předmět:
Molecular biology
Klebsiella pneumoniae
Staphylococcus
Gene Sequencing
Pathology and Laboratory Medicine
Klebsiella Pneumoniae
Feces
Sequencing techniques
Plasmid
Antibiotics
Klebsiella
Medicine and Health Sciences
Staphylococcus Aureus
DNA sequencing
DNA extraction
0303 health sciences
Multidisciplinary
Antimicrobials
Enterobacteriaceae Infections
Drugs
Enterobacteriaceae
Anti-Bacterial Agents
Bacterial Pathogens
R852
Medical Microbiology
Medicine
Pathogens
Research Article
DNA
Bacterial
Science
Biology
Microbiology
03 medical and health sciences
Extraction techniques
Antibiotic resistance
Vancomycin
Culture Techniques
Microbial Control
Drug Resistance
Bacterial
Humans
Microbial Pathogens
030304 developmental biology
Pharmacology
Bacteria
030306 microbiology
Organisms
Computational Biology
Biology and Life Sciences
Sequence Analysis
DNA
biology.organism_classification
R1
Cephalosporins
Resistome
Research and analysis methods
Molecular biology techniques
Metagenomics
Antimicrobial Resistance
Zdroj: PLoS ONE, Vol 14, Iss 11, p e0222831 (2019)
PLoS ONE
ISSN: 1932-6203
1549-1277
Popis: 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.
Databáze: OpenAIRE
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