Characterization of the Urine Microbiome-Host Interaction

• Author / Creator

  Weyant, Robert Benson

• Background: The Human Microbiome is an area of increasing interest, both in and outside of the scientific community. While overall knowledge has increased exponentially, many aspects remain unclear, such as mechanisms of host-interaction and persistence.
  Intracellular bacteria have previously been described in urothelial cells, but only as a mechanism for pathogen persistence in E. coli. Using the urinary microbiome as a model for the human microbiome, we set out to uncover, and better characterize the intracellular microbiome.

  Methods: Participants without recent urinary tract infections or antibiotic use were enrolled in a cross-sectional study. We used several techniques to analyze voided urine samples from each participant to assess for intracellular bacteria. Imaging flow cytometry and volumetric flow cytometry were performed using antibodies against uroplakin III and Enterobacteriaceae common antigen (ECA). Scanning electron microscopy (SEM) also utilized anti-ECA antibody after lysing urothelial cells with sonication. Lastly, 16S rRNA metagenomic sequencing was performed with comparison to a reference library. Pairwise comparisons were conducted using the Wilcoxon rank sum test for continuous variables and the Fisher exact test for proportions. Microbiome analysis using the Qiime2 analysis package with alpha diversity calculated using four different methods: observed richness, Chao1, Shannon index and Simpson index. Beta diversity was calculated using both weighted UniFrac distances and Bray Curtis distances. Differential abundance was calculated using Analysis of Composition of Microbiomes with Bias Correction 2 (ANCOM-BC2).

  Results: We recruited 20 participants of median age 38 (IQR 29-51) with diverse ethnic backgrounds between October-June 2022. Flow cytometry found evidence of intracellular bacteria in 20/20 (100%) participants. Females had a higher number of urothelial cells in their urine (42 cells/μL vs 5 cells/μL, p=0.03) but the proportion of cells containing bacteria was similar at 13.9% (p=0.65). Visualizing with SEM, we found evidence of bacteria in 20/20 (100%) participants. Polymicrobial communities were visualized in a majority of participants (17/20, 85%) and bacteria were aggregated in an extracellular matrix in 20/20 (100%) participants. Metagenomics confirmed the presence of polymicrobial communities in 20/20 (100%) participants and found significant correlation between the concentration of DNA in the intracellular component of urine and the number of urothelial cells containing bacteria (p<0.0001). There were significant differences in alpha diversity, beta diversity, relative abundance, and differential abundance when the extracellular and intracellular urine components were compared.

  Conclusion: We found evidence of a significant intracellular component of the urinary microbiome in healthy individuals. Bacteria were found in 14% of urothelial cells and were visualized together in polymicrobial communities, likely embedded within a biofilm-like structure. The intracellular component was had differing alpha diversity, beta diversity, relative abundance, and difference abundance compared to the extracellular. This large intracellular component of the microbiome explains how the urine microbiome is able to persist within the human urinary tract with its flushing. Sex-based variations were also identified with beta diversity and differential abundance differing significantly between the intracellular components of males and females.
  This is the first-time intracellular bacteria have been described in the urine of healthy individuals. While there are many future implications, this study demonstrates that the method of DNA extraction in microbiome analysis plays a massive role in any potential findings. Future studies on the human microbiome in any location, should evaluate for intracellular bacteria.

• Subjects / Keywords

  • Microbiome
  • Microbiota
  • Urine
  • UTI
  • Intracellular

• Graduation date

  Spring 2024

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-3cn0-kd44

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.