Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications

Abstract

The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia) and some β-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.

Keywords: DNA extraction; Illumina; Nanopore; blood culture broth; host DNA depletion; real-time PCR.

FIG 1

Study workflow. Direct metagenomic sequencing from positive blood culture broth samples was compared with culture-based methods and commercial rapid diagnostics. Two DNA extraction methods were compared, each using half (n = 20) of the samples. AxDx, Accelerate Pheno; BCID, blood culture identification PCR panel (bioMérieux); gDNA, genomic DNA; SPRI, solid-phase reversible immobilization; ID, identification; AST, antimicrobial susceptibility testing; WGS, whole-genome sequencing; MALDI-TOF, matrix-assisted laser desorption ionization–time of flight; AMR, antimicrobial resistance; ML, machine learning; ESBL, extended-spectrum β-lactamase; p-AmpC, plasmid-mediated AmpC β-lactamase.

FIG 2

Comparison of times to results for conventional culture, metagenomic WGS from positive blood culture broth (for short reads), and commercial rapid diagnostic platforms.