Facile Filtration-Based Workflow for Facilitating Sensitive Detection of Pathogenic Bacteria from Blood

Bloodstream infections (BSIs) are a global health concern, contributing to high mortality rates and increased healthcare costs. Current diagnostic methods, such as blood culture, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), immunoassays, and nucleic acid amplification tests (NAATs), have lengthy workflow bottlenecked by the need of culture based method This study introduces an innovative blood processing method that overcomes this limitation, enabling rapid and culture-free isolation of pathogenic bacteria from whole blood. The method combines osmolysis, blood separation membrane filtration, detergent and enzymatic lysis, and bacterial capture, requiring minimal specialized equipment. Notably, it uses a small blood sample volume (0.5 mL or below), making it suitable for pediatric patients. The workflow involves four simple steps and can be completed in approximately 30 minutes, providing rapid pathogen isolation. The protocol was successfully tested with two most common causative bacteria, S. aureus and E. coli, achieving sensitive detection down to approximately 10 CFU using benchtop PCR. The culture-free approach accelerates the diagnosis process and minimizes the risk of bacterial population alterations during culturing. While further optimization and testing with a broader range of pathogens are needed, this method holds promise in advancing the diagnosis of life-threatening bacterial infections. Future applications may include rapid point-of-care testing in resource-limited settings.