%0 Journal Article %A Anson, Luke W. %A Chau, Kevin %A Sanderson, Nicholas %A Hoosdally, Sarah %A Bradley, Phelim %A Iqbal, Zamin %A Phan, Hang %A Foster, Dona %A Oakley, Sarah %A Morgan, Marcus %A Peto, Tim E. A. %A Modernizing Medical Microbiology Informatics Group (MMMIG) %A Crook, Derrick W. %A Pankhurst, Louise J. %T DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing %D 2018 %J Journal of Medical Microbiology, %V 67 %N 3 %P 347-357 %@ 1473-5644 %R https://doi.org/10.1099/jmm.0.000664 %K whole genome sequencing %K sepsis %K bacteraemia %K bloodstream infection %I Microbiology Society, %X 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 63–89 % 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. 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. %U https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.000664