Evaluating Extraction Methods to Study the Canine Urine Microbiome

The urinary microbiome is the collection of microbes present in urine that play a role in host health. Studies of urine microbiota have traditionally relied upon culturing methods aimed at identifying pathogens. However, recent culture-free sequencing studies of the urine microbiome have determined that a diverse array of microbes are present in health and disease. To study these microbes and their potential role in diseases like bladder cancer or interstitial cystitis, consistent extraction and detection of microbial DNA from urine is critical. However, urine is a low biomass substrate. To address this challenge, we tested five different methods for extracting DNA from urine. Urine samples were collected from ten healthy dogs, and 3 ml of urine from each dog was extracted using five different extraction methods including: QIAamp PowerFecal DNA Kit, QIAamp BiOstic Bacteremia DNA Kit, DNeasy Blood & Tissue, QIAamp PowerFecal Pro DNA Kit (Qiagen, Germany), and an extraction protocol using magnetic beads. Extraction methods were compared based on total and bacterial DNA concentrations and microbial community composition and diversity. Significant differences in the urinary microbiota were observed by dog and sex but not extraction method. The Bacteremia kit yielded the highest total DNA concentrations (Kruskal-Wallis, p = 0.165, not significant) and the highest bacterial DNA concentrations (Kruskal-Wallis, p = 0.044). Bacteremia also extracted bacterial DNA from the greatest number of samples. Microbial diversity and composition were not significantly different by kit but did differ significantly by dog and sex. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiome. This work lays the foundation to study the urine microbiome in a wide range urogenital diseases in dogs and other species.