Deciphering antimicrobial resistance in bloodstream infections through clinical metagenomics

BackgroundRapid identification of pathogens and antimicrobial resistance (AMR) in bloodstream infections (BSIs) is critical for timely clinical management. Although blood culture is the reference standard, it is limited by turnaround time and incomplete resolution of resistance mechanisms. We evaluated metagenomic next-generation sequencing (mNGS) applied to flagged positive blood culture bottles to enhance diagnostic resolution and inform targeted molecular approaches. MethodsFifty-five flagged positive blood culture bottles from a tertiary care hospital in Bengaluru, India, were analyzed. Shotgun mNGS was performed directly on blood culture broth and compared with routine phenotypic identification and antimicrobial susceptibility testing (AST) from corresponding isolates. Antimicrobial resistance genes (ARGs) and plasmid replicons were profiled. ResultsmNGS showed high concordance with routine culture for pathogen identification (54/55; 98.2%) and improved species-level resolution across bacterial and fungal pathogens. Genotypic resistance profiles were consistent with phenotypic AST, identifying {beta}-lactamases, efflux-associated determinants, and target modification mechanisms. Diverse ARGs and plasmid replicons (Inc-, Col-, and rep-family) were detected, providing genomic context for resistance. Sequencing predominantly reflected the cultured organism, supporting high specificity in flagged blood culture material. ConclusionsmNGS applied to flagged blood culture bottles enables high-resolution characterization of pathogens and resistance determinants at a clinically actionable stage. The genomic insights generated provide a framework for developing targeted multiplex PCR assays that can reduce turnaround time and improve affordability compared with sequencing-based approaches. This strategy supports the use of mNGS as an adjunct to conventional diagnostics and as a bridge toward scalable, rapid, and cost-effective solutions for BSI diagnosis and AMR surveillance.