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High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure,  Y. pestis ,  F. tularensis  and  B. anthracis  spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5–5.9 orders of magnitude, in samples with bacteria spiked-in at 10 3 –10 5  CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8–9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample.

A rapid high-throughput sequencing-based approach for the identification of unknown bacterial pathogens in whole blood