DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing

• Published in: Journal of medical microbiology Vol. 67; no. 3; pp. 347 - 357
• Main Authors: Anson, Luke W, Chau, Kevin, Sanderson, Nicholas, Hoosdally, Sarah, Bradley, Phelim, Iqbal, Zamin, Phan, Hang, Foster, Dona, Oakley, Sarah, Morgan, Marcus, Peto, Tim E A, Modernizing Medical Microbiology Informatics Group Mmmig, Crook, Derrick W, Pankhurst, Louise J
• Format: Journal Article
• Language: English
• Published: England Microbiology Society 01.03.2018
• Subjects: Bacteremia - diagnosis; Bacteremia - microbiology; Blood Culture; Catheter-Related Infections - diagnosis; Catheter-Related Infections - microbiology; DNA, Bacterial - analysis; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Escherichia coli - genetics; Genome, Bacterial; Humans; Microbial Sensitivity Tests; Molecular Diagnostic Techniques - methods; Reagent Kits, Diagnostic; Sequence Analysis, DNA - methods; Staphylococcus aureus - genetics; Whole Genome Sequencing; whole genome sequencing; bloodstream infection; sepsis; bacteraemia
• ISSN: 0022-2615; 1473-5644
• DOI: 10.1099/jmm.0.000664

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. 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 , versus 0-2.5 ng µl using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9-2.1) and human DNA (0.0-4.4×10  copies) in BiOstic extracts. Differential centrifugation (2000 g, 1 min) prior to BiOstic extraction reduces human DNA by 63-89 % with selective lysis minimizing by a further 62 %. Post-extraction bead clean-up lowers inhibition. Overall, 67 % of sequenced samples (Illumina MiSeq) contain <10 % human DNA, with >93 % concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If >60 % 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. Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay.