Alterations of microbiota in urine from women with interstitial cystitis

Abstract

Background: Interstitial Cystitis (IC) is a chronic inflammatory condition of the bladder with unknown etiology. The aim of this study was to characterize the microbial community present in the urine from IC female patients by 454 high throughput sequencing of the 16S variable regions V1V2 and V6. The taxonomical composition, richness and diversity of the IC microbiota were determined and compared to the microbial profile of asymptomatic healthy female (HF) urine.

Results: The composition and distribution of bacterial sequences differed between the urine microbiota of IC patients and HFs. Reduced sequence richness and diversity were found in IC patient urine, and a significant difference in the community structure of IC urine in relation to HF urine was observed. More than 90% of the IC sequence reads were identified as belonging to the bacterial genus Lactobacillus, a marked increase compared to 60% in HF urine.

Conclusion: The 16S rDNA sequence data demonstrates a shift in the composition of the bacterial community in IC urine. The reduced microbial diversity and richness is accompanied by a higher abundance of the bacterial genus Lactobacillus, compared to HF urine. This study demonstrates that high throughput sequencing analysis of urine microbiota in IC patients is a powerful tool towards a better understanding of this enigmatic disease.

Figure 1

Summary of the microbial phyla and orders detected in interstitial cystitis urine and healthy female urine. A: A comparative taxonomic tree view of 16S rDNA sequences from interstitial cystitis (IC) urine and healthy female (HF) urine assigned to the phylum level as computed using MEGAN V3.4. Normalized counts by pooling together results from V1V2 and V6 16S rDNA sequence datasets were used for both IC and HF urine. B and C: Comparison of taxonomic assignments for IC and HF urine sequences at the order level, showing an increase of the order Lactobacillales in IC urine sequences relative to HF urine, for both V1V2 (B) and V6 datasets (C).

Figure 2

Hierarchical clustering of urine microbiomes. Heat map showing the relative abundance of bacterial genera across the urine samples. Genera are listed to the right. Subjects are listed at the top: interstitial cystitis (IC) samples denoted as P_number_V1V2 or V6, and healthy female (HF) urine samples as F_number_V1V2 or V6. Pink indicates IC urine, green HF urine. Color intensity of the heat map is directly proportional to log 10 scale of the abundance normalized sequence data as done by MEGAN V3.4. Taxa marked with (*) are genera that were significantly (p ≤ 0.05, p value from Metastats) different between the IC and HF urine microbiota. Genera marked with (†) and (§) are unique for HF urine sequences and IC urine sequences, respectively. Note that most of the IC urine samples are less complex than what is seen for HF urine samples.

Figure 3

Comparison of richness and diversity estimations of urine from interstitial cystitis (IC) patients and healthy females (HF). A: Rarefaction curves depicting number of OTUs (at 3% genetic difference) as function of the total number of sequences for the combined sequence pool datasets for IC urine V1V2 and V6 (red and orange) and HF urine V1V2 and V6 (dark and light blue). The curves show a decreased estimate of species richness in the IC urine microbiome compared to the HF urine microbiome. B, C, and D: Box plots showing richness and diversity of 16S rDNA sequences. Boxes contain 50% of the data and have lines at the lower quartile (red), median and upper quartile (green) values. Ends of the whiskers mark the lowest and highest value. The plots show the results of a combined assessment of the eight urine samples in each HF and IC microbiome and with normalized numbers of sequences for OTU and Shannon index values (B and C). B: Observed OTU counts (at 3% genetic difference) of all urine samples taken from HF and IC, for both V1V2 and V6 datasets. C and D: Shannon index and inverse Simpson index at 3% sequence dissimilarity calculated to estimate diversity for both V1V2 and V6 datasets. Asterisks (*) indicate significant differences (Wilcox rank sum test: * p < 0.05). Note that a single sample (P2) in the IC community is the only outlier with the highest values for both richness and diversity (for both V1V2 and V6 analysis).

Figure 4

OTU based clustering analysis of urine microbiomes. Non-metric multidimensional scaling (NMDS) plots were generated based on θYC distances (0.03) between interstitial cystitis (IC) and healthy female (HF) microbiomes for both V1V2 (A) and V6 region (B). Red: IC patient samples; blue: HF samples.