Development and optimization of host DNA depletion in blood cultures using a saponin and salt-activated nuclease-based method

Early diagnosis of sepsis and bloodstream infections is essential to reduce mortality and combat antimicrobial resistance. However, current diagnostic methods are slow and laborious. Nanopore sequencing has the potential to be a rapid diagnostic tool for sepsis. Nonetheless, extracting bacterial DNA from blood samples remains challenging due to the abundance of host DNA and the presence of inhibitory substances. This study aimed to develop and optimize a method using saponin and salt-activated nucleases (SAN) for efficient host DNA depletion in blood cultures. Different saponin concentrations (2%–5%), HL-SAN and M-SAN nucleases, and various salt conditions (NaCl and MgCl 2 ) were tested for their ability to reduce host DNA in Escherichia coli and Staphylococcus aureus blood cultures. The effects of bead-beating durations of 6 and 10 min on DNA fragment size distribution were also evaluated. The optimized method was validated using spiked and clinical blood cultures of the most prevalent BSI causing pathogens. Results showed that 4% of saponin treatment combined with 250 units (10 μL) of SAN nucleases achieved efficient host DNA depletion in spiked blood cultures. Compared to undepleted samples, HL-SAN achieved up to 38-fold reduction in host DNA for both E. coli and S. aureus . Likewise, M-SAN resulted in ~10-fold reduction in E. coli and ~4-fold reduction in S. aureus samples. Although SAN enzymes combined with higher salt concentrations showed slightly better host DNA removal, the difference was not statistically significant. Shortening the bead-beating time to 6 min improved the recovery of longer DNA fragments compared to 10 min. The optimized method validated using spiked and clinical-positive blood cultures, demonstrated efficient host depletion and bacterial DNA recovery.