Rapid and modular workflows for same-day sequencing-based detection of bloodstream infections and antimicrobial resistance determinants using culture-enriched samples

Bloodstream infections (BSI) are a major global health concern, and existing diagnostic methods are too slow to guide targeted antibiotic therapy for critically ill patients. Rapid metagenomic next-generation sequencing (mNGS) can facilitate swift microbiological diagnosis, but identification is challenged by significant host versus bacterial DNA in blood and blood culture media. To accelerate reporting time, we developed M-15, a rapid mNGS-based host DNA depletion workflow optimized for culture-enriched samples, validated with suspected BSI blood culture samples and rapid culture-enriched spiked blood. M-15 was benchmarked with five commercial/published protocols, combined with rapid mNGS, and tested on blood culture samples ( n = 33) from suspected BSI cases identified on BACT/ALERT-VIRTUO. To determine whether it is possible to utilize M-15 mNGS prior to blood culture flagging positive, a rapid enrichment method was tested starting with 1–10 colony-forming units of the top 15 bacterial species causing BSI spiked into BACT/ALERT medium enriched with 10 mL sheep blood. All six chemical depletion protocols reduced host DNA by 2.5 × 10 0 - to 4.1 × 10 6 -fold, with the in-house M-15 protocol performing best. With BACT/ALERT specimens, M-15 mNGS identified 28/28 mono-bacterial and 2/4 multi-bacterial species. With rapid culture enrichment and M-15 mNGS, <18% DNA was classified as host, and all bacterial species tested ( n = 10) were correctly identified. M-15 mNGS accurately predicted phenotypic AMR/susceptibility for 90.3% (232/257) of drug/bacteria combinations from BACT/ALERT-positive samples. This study demonstrates that M-15 mNGS can facilitate species and AMR gene detection within 5–7 hours of BACT/ALERT positivity and possibly 13–15 hours of sample collection. Further clinical validation is required to assess its performance and the potential to improve patient outcomes in BSI. IMPORTANCE Bloodstream infections (BSI) are among the leading global health challenges, and traditional culture-based diagnostic methods are too slow (often taking >48 hours) to guide critical clinical interventions. This study demonstrates the development and utility of M-15 metagenomic next-generation sequencing (mNGS), a modular Oxford Nanopore-based chemical host DNA depletion and metagenomic sequencing workflow applied to enriched blood culture media for the same-day detection of bacterial etiologies and their antimicrobial resistance (AMR) genes. The selective chemical host DNA depletion method (M-15) described in this study can remove approximately 4.1 × 10 6 -fold unwanted host DNA from whole blood, providing high-resolution genomic information from the bacteria at a fraction of the sequencing time/cost (approximately £120–£160/sample). We have tested this workflow on culture-positive clinical and rapid enriched spiked blood samples and demonstrated its ability to identify bacterial species and AMR genes between 5 and 7 hours post blood culture positivity. Based on our in vitro experiments using rapid enrichment, we believe similar results could be achieved within 13–15 hours from blood sample collection. Although further clinical validation is required, especially to fully assess the rapid version of the protocol, M-15 mNGS offers a promising advancement in BSI diagnosis. This workflow is modular and can be expanded in the future to adapt for other infections, which makes it a versatile tool to improve patient outcomes in sepsis.