Sequential co-extraction of gut microbial DNA and fecal polyamines enables integrated microbiome-metabolite profiling...

Early, non-invasive biomarkers for Alzheimer's disease (AD) are urgently needed. Impaired polyamine metabolism, regulated by intracellular pathways and the gut microbiota, has been reported in postmortem brains of AD patients. Here, we developed a sequential co-extraction workflow (Method 10) that enables recovery of gut bacterial DNA and fecal free polyamines-putrescine, spermidine, and spermine-from the same small mouse fecal sample, allowing paired microbiome and metabolite profiling. Applying this workflow to an AD knock-in mouse model and age-matched controls at 8, 32, and 56 weeks revealed an early decrease in Lactobacillus abundance at 8 weeks accompanied by elevated spermidine levels (p < 0.05), while total fecal polyamine concentrations increased further in AD mice at 56 weeks. These findings suggest that integrated fecal microbiome-polyamine profiling may provide exploratory microbiota-polyamine signatures associated with AD progression.