Abstract
The urinary microbiota is the collection of microbes present in urine that may 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 is 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 bacterial 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 bacterial 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.
Highlights
Urine, in the absence of urinary tract infection, has long been considered sterile; a principle still taught in many healthcare professional settings to date
The number of samples with quantifiable DNA varied by extraction method (Fig 1A)
No DNA was recovered from the spiked positive control sample using PowerFecal1 Pro (PFP)
Summary
In the absence of urinary tract infection, has long been considered sterile; a principle still taught in many healthcare professional settings to date. Evidence counter to this idea has been accumulating for several decades [1]. Culture-positive asymptomatic bacteriuria is commonly reported in women and older adults; this is sometimes deemed “contamination” based on bacterial counts < 105 [2]. Culture-negative symptomatic urinary tract infections (UTIs) are common, and can, in some cases, be attributed to fastidious organisms that fail to grow using standard urine culturing (SUC) techniques [2,3,4,5,6,7,8]. Culture-independent next-generation sequencing of urine and enhanced quantitative urine.
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