Evaluating extraction methods to study canine urine microbiota

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.

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

“…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.…”

Determination of the Optimal Bacterial DNA Extraction Method to Explore the Urinary Microbiota

“…Illustrating the importance of the extraction step in an analytical process, two very recent studies have also benchmarked DNA extraction kits for the study of human and canine urine microbiota [20,24]. Mrofchak et al identified the BI kit as having the best extraction method for the study of the urine microbiota, whereas the study by Karstens et al reported that the BI and BT kits gave similar performances.…”

Urine and Fecal Microbiota in a Canine Model of Bladder Cancer

“…Urine samples were extracted using QIAamp ® BiOstic ® Bacteremia DNA Isolation Kit (Qiagen, Hilden, Germany) as described previously [68]. Fecal samples were extracted using the QIAamp ® PowerFecal ® DNA Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.…”

“…Likewise, negative controls underwent the full extraction, library preparation, and sequencing process. We amplified the V4 region of the 16S rRNA gene using primers 515F and 806R, and PCR and sequencing were performed as described previously (2 x 250bp paired-end reads, on an Illumina Miseq (Lemont, IL, USA)) [68][69][70]. Raw, paired-end sequence reads were processed using QIIME2 v. 2020.11 and DADA2 [71,72].…”