Abstract
Bloodstream infection is a major cause of morbidity and mortality worldwide. We explored whether MinION nanopore sequencing could accelerate diagnosis, resistance, and virulence profiling prediction in simulated blood samples and blood cultures. One milliliter of healthy blood samples each from direct spike (sample 1), anaerobic (sample 2), and aerobic (sample 3) blood cultures with initial inoculation of ∼30 CFU/ml of a clinically isolated Klebsiella pneumoniae strain was subjected to DNA extraction and nanopore sequencing. Hybrid assembly of Illumina and nanopore reads from pure colonies of the isolate (sample 4) was used as a reference for comparison. Hybrid assembly of the reference genome identified a total of 39 antibiotic resistance genes and 77 virulence genes through alignment with the CARD and VFDB databases. Nanopore correctly detected K. pneumoniae in all three blood samples. The fastest identification was achieved within 8 h from specimen to result in sample 1 without blood culture. However, direct sequencing in sample 1 only identified seven resistance genes (20.6%) but 28 genes in samples 2–4 (82.4%) compared to the reference within 2 h of sequencing time. Similarly, 11 (14.3%) and 74 (96.1%) of the virulence genes were detected in samples 1 and 2–4 within 2 h of sequencing time, respectively. Direct nanopore sequencing from positive blood cultures allowed comprehensive pathogen identification, resistance, and virulence genes prediction within 2 h, which shows its promising use in point-of-care clinical settings.
Highlights
Bloodstream infections (BSIs) are a major cause of morbidity and mortality worldwide
Species identification and antibiotic resistance genes and virulence genes analyses were performed based on real-time nanopore sequencing data (Figure 1B)
We sought to demonstrate the potential of the nanopore instrument for metagenomic pathogen identification, as well as antimicrobial drug resistance and virulence genes prediction directly from simulated BSI samples
Summary
Bloodstream infections (BSIs) are a major cause of morbidity and mortality worldwide. The identification of a pathogen and subsequent antimicrobial susceptibility testing rely largely on the microbial growth, which may take at least 1 day to months (Grumaz et al, 2016; Anson et al, 2018; Ashikawa et al, 2018). This culture-based procedure may yield false-negative results if the patient is given empiric antibiotic therapy before BC is performed (Grumaz et al, 2016). Early identification of the causative pathogen as well as its antibiotic resistance pattern is the highest priority for improving patient prognosis
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