Impact of sample processing method and volume on 16 S rRNA profiling of the urobiome

BACKGROUND: The urinary microbiome (urobiome) plays important roles in both human and animal urogenital tract health. Characterization of these microbial communities presents several technical challenges, largely due to the low microbial biomass of urine. Whereas other low biomass liquid systems, such as aquatic samples, frequently employ small-pore vacuum filtration for microbial DNA concentration, urobiome studies have traditionally relied on centrifugation and pelleting of smaller volumes. Therefore, this study compared the effects of processing method (vacuum filtration versus pelleting) and sample volume on bacterial DNA yield, contaminant burden, and microbial diversity in canine urine. CONCLUSIONS: These findings indicate that interindividual variation predominates over methodological effects. Higher urine volumes (≥ 30 mL) were associated with technical challenges in filtered samples, whereas moderate urine volumes (1-10 mL) appear sufficient for urobiome characterization. Similar microbial recovery, increased contaminant signal, and occasional clogging with filtration suggests that pelleting remains an appropriate approach for urobiome characterization.