Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic urological condition diagnosed in nearly 8 million women in the United States. Whether the urinary microbiota plays an etiologic role remains controversial. Most studies have assessed the microbiota of IC/BPS patients with voided or catheterized urine as a proxy for bladder urothelium and, although urine is a convenience sample, it may not be a true reflection of the bladder microbiota. The use of bladder biopsy tissue may provide a more accurate, and thus more clinically relevant, picture of the bladder microbiota.

Bladder biopsy tissues were obtained from: (a) 30 women with IC/BPS (18-80 y/o) via cystoscopically guided cold-cup biopsy following therapeutic bladder hydrodistension, and (b) 10 non-IC/BPS women undergoing pelvic organ prolapse repair. To detect bacteria, technical duplicates of each RNAlater-preserved biopsy were subjected to amplicon sequencing of the V4 region of the 16S rRNA gene. To visualize bacteria, paraformaldehyde-fixed, paraffin-embedded biopsies were subjected to combined fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) using confocal microscopy.

Bacteria were detected in at least one technical replicate of every biopsy (Fig 1). The most abundant genus was Staphylococcus (light blue), followed by Lactobacillus (dark blue); Escherichia (gold) was common but not abundant. Other common but not abundant genera include Gardnerella (maroon), Pseudomonas (light green) and Mycoplasma (light purple). Combined FISH/IHC reproducibly detected perinuclear clusters of 16S RNA in epithelial cells of human bladder biopsy tissue (Fig 2). There was no significant difference between IC/BPS patients and controls (p>0.05).

We detected and visualized bacteria associated with the bladder urothelium in 40 women. The urothelial microbiota are similar but not identical to the urinary microbiota reproducibly observed in multiple earlier studies (e.g., Lactobacillus is more abundant in urine; Staphylococcus is more abundant in the urothelium). These results are consistent with the hypothesis that bacteria are associated with the urothelium, that they are sloughed off into the bladder lumen, and that some genera more easily shed into the bladder lumen than others. The perinuclear location of the visualized bacteria suggest that they reside within the urothelial cells. This is consistent with previous reports that some bacteria are internalized by urothelial cells.

We conclude that bacteria are associated with the urothelium. We further conclude that these bacteria are likely within the urothelial cells. We did not find evidence that urothelial microbiota differs between women with IC/BPS and non-IC/BPS controls but this should not be taken as definitive; much larger studies are indicated.