Characterization of the urinary microbiome in healthy dogs

Abstract

The urinary bladder in healthy dogs has dogmatically been considered free of bacteria. This study used culture independent techniques to characterize the healthy canine urinary microbiota. Urine samples collected by antepubic cystocentesis from dogs without urinary infection were used for DNA extraction. Genital tract and rectal samples were collected simultaneously from the same dogs. The V4 hypervariable region of the 16S rRNA bacterial gene was amplified and compared against Greengenes database for OTU assignment and relative abundance for urine, genital, and rectal samples. After excluding 4 dogs with cultivable bacteria, samples from 10 male (M; 1 intact) and 10 female (F) spayed dogs remained. All samples provided adequate genetic material for analysis. Four taxa (Pseudomonas sp., Acinetobacter sp., Sphingobium sp. and Bradyrhizobiaceae) dominated the urinary microbiota in all dogs of both sexes. These taxa were also detected in the genital swabs of both sexes, while the rectal microbiota differed substantially from the other sample sites. Principal component (PC) analysis of PC1 through PC3 showed overlap of urinary and genital microbiota and a clear separation of rectal swabs from the other sample sites along PC1, which explained 44.94% variation. Surprisingly, the urinary microbiota (mean # OTU 92.6 F, 90.2 M) was significantly richer than the genital (67.8 F, 66.6 M) or rectal microbiota (68.3 F, 71.2 M) (p < 0.0001), with no difference between sexes at any sample site. The canine urinary bladder is not a sterile environment and possesses its own unique and diverse microbiota compared to the rectal and genital microbiota. There was no difference between the sexes at any microbiota sample site (urine, genital, and rectal). The predominant bacterial genus for either sex in the urine and genital tracts was Pseudomonas sp.

Fig 1

Dot plots showing Chao1 indices (A), Shannon indices (B), and operational taxonomic unit (OTU) richness (C) of microbiota detected in the urine samples, genital swabs, and rectal swabs, collected from 20 healthy female (F, n = 10) and male (M, n = 10) dogs; bar chart showing the number of OTUs detected in X out of 20 urine samples, overlaid with dots indicating the mean relative abundance of those OTUs, demonstrating the high sparsity of the urinary microbiota. *p<0.05, **p<0.01, ***p<0.001.

Fig 2. Stacked bar charts showing relative abundance of microbial DNA detected via 16S rRNA amplicon sequencing and annotated to the taxonomic level of family, in samples collected via cystocentesis (urine), vaginal or preputial swab (genital swab), or rectal swab from 20 healthy adult dogs (n = 10 female, 10 male).

Fig 3. Principal component analysis of urine, genital swab, and rectal swab microbiota, as determined via 16S rRNA amplicon sequencing, colored by sample collection site, from 20 healthy adult dogs (n = 10 female, 10 male).

Plots depict PC1 versus PC2 (A), PC1 versus PC3 (B), and PC1 versus PC4 (C).

Fig 4. Agglomerative hierarchical clustering analysis of Bray-Curtis similarity indices, performed via unweighted pair group method with arithmetic mean (UPGMA), legend at right.