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.HighlightsCanine urine microbiota differed by sex and dog but not extraction method.Qiagen Bacteremia kit yielded the highest bacterial DNA concentrations from urine.The Bacteremia kit extracted bacterial DNA from the greatest number of samples.Absolute abundance of Sphingomonas species increased in female dog urine.Pasteurellaceae bacterium canine oral taxon 272 increased in male dog urine.

Characterization of the urogenital microbiome in Miniature Schnauzers with and without calcium oxalate urolithiasis

“…Thus, whether true pathogenic species or strains were present remains unclear. However, this finding is consistent with previous reports in dogs 19‐21 and highlights the complexity of defining infectious pathogens given emerging awareness of commensal urinary microbes.…”

“…Taxonomic profiles in the study urine samples were dominated by members of the Proteobacteria phylum, which is consistent with previous findings in both dogs and cats 19,20,60 . In contrast, the genera Lactobacillus and Gardnerella commonly dominate urotypes of women 25,61,62 .…”

“…A preservative was not used in our study. Although other urine and urogenital microbiome studies in dogs and cats have been performed without such preservatives, 19,20,60 it is unknown how the lack of preservative affected the results of our study. Careful sample selection, uniform processing of the specimens, and stringent bioinformatic removal of putative contaminants were employed to mitigate technical biases.…”

“…Taxonomic profiles in the study urine samples were dominated by members of the Proteobacteria phylum, which is consistent with previous findings in both dogs and cats. 19,20,60 In contrast, the genera Lactobacillus and Gardnerella commonly dominate urotypes of women. 25,61,62 These differences might be a result of variations in urine collection method, anatomic or hormonal differences between species, or other biological or environmental factors.…”

“…9,10,18 In fact, a comprehensive analysis of the gut and urogenital microbiomes determined that the urogenital microbiome exhibited more pronounced alterations by urolith status than did the gut microbiome. 9 Microbial communities also recently have been identified in the urine of healthy dogs, [19][20][21] but the role of these microbes in CaOx formation in dogs is unknown. Discovery of urogenital microbiome features that discriminate dogs with CaOx urolithiasis from healthy dogs could provide insight into disease pathogenesis.…”