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

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.