Rapid Identification of Aerobic Growth in Sepsis Pathogens by Flow Cytometry 2254627

Abstract Introduction Sepsis is a life-threatening syndrome with 30% mortality in severe cases. Early therapy within 6 hours improves prognosis by 50%, but conventional identification by blood culture and subsequent analyses requires ≥24 h. During this delay, broad-spectrum antibiotics are used, and prolonged administration promotes antimicrobial resistance, emphasizing the need for faster growth characterization methods. We aimed to establish flow cytometry as a tool for rapid assessment of aerobic proliferation. Specifically, we examined whether scatter-based analysis could discriminate aerobic from non-aerobic bacterial strains within 6 hours, across inoculum sizes. We also assessed detection sensitivity, reproducibility of scatter metrics, and temporal changes in bacterial distributions to provide experimental evidence supporting early pathogen discrimination. Methods Two bacterial strains known to proliferate aerobically and three that do not were adjusted to 1, 10, 10², and 10³ CFU/mL. Samples were incubated aerobically and analyzed with the UF-5000 system at 0, 1, 2, 3, and 6 hours. Growth kinetics were quantified, and forward scatter and side scatter distributions were evaluated. The area encompassing 95% of bacterial events was calculated. Results Enterococcus faecalis and Escherichia coli became detectable within 6 hours at ≥ 10 CFU/mL. Their bacterial counts rose progressively after 3 hours, with clear proliferation by 6 hours. Scatter plots showed expansion of the 95% event area to > 50 times of baseline at 6 hours. Non-aerobic strains (Bacteroides fragilis, Prevotella melaninogenica and Clostridium perfringens) remained undetectable even after 6 hours at 10³ CFU/mL and showed no significant change in scatter distribution. Conclusion Flow cytometry enables fundamental assessment of aerobic growth capacity within 3 hours. Differences in bacterial proliferation under aerobic conditions can be detected at an early stage, offering a basis for experimental discrimination of sepsis pathogens. Funding Source JSPS KAKENHI, JP 24K11096 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)