DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing.
J Med Microbiol
; 67(3): 347-357, 2018 Mar.
Article
en En
| MEDLINE
| ID: mdl-29458686
ABSTRACT
PURPOSE:
Speed of bloodstream infection diagnosis is vital to reduce morbidity and mortality. Whole genome sequencing (WGS) performed directly from liquid blood culture could provide single-assay species and antibiotic susceptibility prediction; however, high inhibitor and human cell/DNA concentrations limit pathogen recovery. We develop a method for the preparation of bacterial DNA for WGS-based diagnostics direct from liquid blood culture.METHODOLOGY:
We evaluate three commercial DNA extraction kits BiOstic Bacteraemia, Amplex Hyplex and MolYsis Plus. Differential centrifugation, filtration, selective lysis and solid-phase reversible immobilization bead clean-up are tested to improve human cells/DNA and inhibitor removal. Using WGS (Illumina/MinION), we assess human DNA removal, pathogen recovery, and predict species and antibiotic susceptibility inpositive blood cultures of 44 Gram-negative and 54 Staphylococcus species.Results/Key findings. BiOstic kit extractions yield the greatest mean DNA concentration, 94-301 ng µl-1, versus 0-2.5 ng µl-1 using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9-2.1) and human DNA (0.0-4.4×106 copies) in BiOstic extracts. Differential centrifugation (2000 g, 1 min) prior to BiOstic extraction reduces human DNA by 63-89â% with selective lysis minimizing by a further 62â%. Post-extraction bead clean-up lowers inhibition. Overall, 67â% of sequenced samples (Illumina MiSeq) contain <10â% human DNA, with >93â% concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If >60â% of sequencing reads are human (7/98 samples) susceptibility prediction becomes compromised. Novel MinION-based WGS (n=9) currently gives rapid species identification but not susceptibility prediction.CONCLUSION:
Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay.Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN Bacteriano
/
Genoma Bacteriano
/
Bacteriemia
/
Cultivo de Sangre
/
Secuenciación Completa del Genoma
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Med Microbiol
Año:
2018
Tipo del documento:
Article
País de afiliación:
Reino Unido