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

Background 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, risking poor clinical outcomes. Rapid metagenomic-sequencing (mNGS) can facilitate swift pathogen and antimicrobial resistance (AMR) detection, but identification is challenged by significant host versus bacterial DNA in blood. To accelerate microbiological diagnosis, we developed M-15, a rapid and modular mNGS-based host DNA depletion workflow, validated with suspected BSI blood-culture samples and rapid culture-enriched spiked blood. Methods To assess chemical host DNA depletion (CHDD) efficiency, M-15 was benchmarked with five commercial/published protocols. Later, M-15 was combined with rapid mNGS with/without adaptive sampling (AS) and tested on clinical blood-culture samples (n = 33) from suspected BSI cases identified on BACT/ALERT VIRTUO (30 flagged positive, three remained negative). To determine whether it is possible to utilise 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 BACTEC medium enriched with 10 mL sheep blood. Results All six chemical depletion protocols reduced host DNA by 2.5x10 ⁰ to 4.1x10 ⁶ -fold, with the in-house M-15 protocol performing best, while adaptive sampling depleted host > 5-fold. With BACT/ALERT specimens, M-15 mNGS accurately identified 3/3 negative, 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. Conclusions This study demonstrates that M-15 mNGS can facilitate species and AMR gene detection within 5–7 hours of BACT/ALERT positivity. Including 8-hour culture enrichment, microbiological and AMR confirmation is possible within 13–15 hours of sample collection. Thus, the M-15 mNGS workflow has the potential to improve patient outcomes in BSI.