Bacterial cell-free DNA profiling reveals the co-elevation of multiple bacteria in newborn foals with suspected sepsis.

Sepsis is the leading cause of death in neonatal foals, yet current diagnostics lack sufficient sensitivity and specificity. Here, we present a foal cell-free DNA (cfDNA) sequencing for bacterial identification (cfFBI) workflow that integrates wet-lab and computational protocols, enabling direct bacterial profiling through enrichment of the bacterial cfDNA and minimization of false-positive detections. We applied cfFBI to blood from 25 hospitalized foals and 7 healthy foals (H). Sepsis-associated bacterial genera were elevated in all 11 nSIRS-positive (S+) foals compared to H, and in 8/11 when compared to both nSIRS-negative (nS-) and H, with multiple genera elevated in nearly half (45.5%). While total cfDNA concentration, bacterial fraction, and microbial diversity did not differ between groups, S+ foals showed distinct cfDNA end-motif patterns and reduced mitochondrial cfDNA fractions. These findings indicate that cfDNA sequencing enables the detection of pathogenic bacteria and can help identify additional (host-related) sepsis biomarkers.