A simple, low-cost, and highly efficient protocol for rapid isolation of pathogenic bacteria from human blood.

Bacteremia is a serious clinical condition in which pathogenic bacteria enter the bloodstream, putting patients at risk of septic shock and necessitating aggressive antibiotic treatment. Choosing the most effective antibiotic is crucial not only for resolving the infection but also for minimizing side effects, such as dysbiosis in the healthy microbiome and mitigating the evolution of antibiotic resistance. This requires rapid identification of the pathogen and antibiotic susceptibility testing, yet these processes are inherently slow in standard clinical microbiology labs due to reliance on growth-based assays. Although alternative methods exist, they are rarely adopted in clinical settings because they involve complex protocols and high costs for retraining the personnel and new equipment. Here, we present an optimized and straightforward protocol for the rapid and efficient isolation of bacterial pathogens directly from blood samples, without disrupting standard laboratory workflows. This cost-effective approach utilizes commonly available laboratory equipment and enables direct bacterial cell isolation. By eliminating the need for traditional blood culture steps, it significantly reduces diagnostic delays while remaining fully compatible with downstream bacterial identification analyses. Our protocol achieves over 70% bacteria isolation efficiency within 30 min, remained effective at low bacterial concentrations (1-10 bacteria/0.3 mL blood), and preserved bacterial viability with no notable change in growth lag times. We validated the protocol on several clinically relevant bacterial species, including