Akira Asai, MD PhD: No financial relationships to disclose
Introduction/Rationale: 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.
