MultiSeq-AMR: A modular amplicon-sequencing workflow for rapid detection of bloodstream infection and antimicrobial resistance markers

Bloodstream infections (BSI) represent a significant global health challenge, and traditional diagnostic methods are suboptimal for timely guiding targeted antibiotic therapy. We introduce MultiSeq-AMR, an ultra-fast amplicon sequencing workflow to identify bacterial and fungal species, and a comprehensive set of antimicrobial resistance (AMR) genes (n = 91) for BSI diagnosis. We initially benchmarked MultiSeq-AMR using DNA from 21 bacterial and fungal isolates and accurately identified 100% species and 99.4% AMR genes. Further validation with 33 BACT/ALERT positive samples from suspected BSI cases revealed 100% accuracy for pathogen identification with mono-bacterial samples, with 97.4% categorical agreement (CA) for AMR gene prediction. To accelerate diagnosis, 6-hour culture-enrichment combined with MultiSeq-AMR identified 11/13 species with 96% CA for AMR gene identification. MultiSeq-AMR holds promise for improving patient outcomes as species/AMR genes could be identified in under 5 hours of BACT/ALERT positivity, and potentially <11 hours of sample collection.