Evaluating Extraction Methods to Study Canine Urine Microbiota

Abstract

The urinary microbiota 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 microbiota 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, requiring sensitive methods to capture DNA and making the risk of contamination high. To address this challenge, we collected urine samples from ten healthy dogs and extracted DNA from each sample using five different commercially available extraction methods. Extraction methods were compared based on total and bacterial DNA concentrations and microbial community composition and diversity assessed through 16S rRNA gene sequencing. 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. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiota. This work lays the foundation to study the urine microbiome in a wide range of urogenital diseases in dogs and other species. HighlightsO_LICanine urine microbiota differed by sex and dog but not extraction method. C_LIO_LIQiagen Bacteremia kit yielded the highest bacterial DNA concentrations from urine. C_LIO_LIThe Bacteremia kit extracted bacterial DNA from the greatest number of samples. C_LIO_LIAbsolute abundance of Sphingomonas species increased in female dog urine. C_LIO_LIPasteurellaceae bacterium canine oral taxon 272 increased in male dog urine. C_LI