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Urobiome: State of Knowledge, and Evaluation of Methods for Enabling Genome-Resolved Metagenomics of Urinary Tract Microbiota

Lewis, Zachary Jordan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1720651049588034

Abstract Details

2024, Master of Science, Ohio State University, Veterinary Preventive Medicine.
The urinary tract contains a distinct and diverse set of microbiota (urobiome). Important work has established links between the composition of the urobiome and various diseases of the urinary tract, including bladder cancer, urolithiasis, incontinence, and recurrent urinary tract infection. However, efforts to characterize the urobiome and assess its functional potential have been limited due to technical challenges including low microbial biomass and high host cell shedding in urine. To begin addressing these challenges, we evaluate urine sample volume (100 l – 5 mL), and host DNA depletion methods and their effects on urobiome profiles in healthy dogs, which are a robust large animal model for the human urobiome. We collected urine from seven dogs and fractionated samples into multiple aliquots. One set of samples was additionally spiked with host (canine) cells to model a biologically relevant host cell burden in urine. Samples then underwent DNA extraction followed by 16S and shotgun metagenomic sequencing. We tested six methods of DNA extraction: QIAamp BiOstic Bacteremia (no host depletion), QIAamp DNA Microbiome, Molzym MolYsis, NEBNext Microbiome DNA Enrichment, Zymo HostZERO, and Propidium Monoazide. Sequences were processed and analyzed using QIIME2 and MetaPhlAn4. Metagenome assembled genomes (MAGs) were generated using MEGAHIT and MetaWRAP pipelines. Statistical analysis were performed in R. In relation to urine sample volume,  3.0 mL resulted in the most consistent urobiome profiling. In relation to host depletion, individual (dog) but not extraction method drove overall differences in microbial composition. DNA Microbiome yielded the greatest microbial diversity in 16S (p=0.0025) and shotgun metagenomic data (p=0.01), and maximized MAG recovery while effectively depleting host DNA (p=0.0039) in host-spiked urine samples. As proof-of-principle, we then mined MAGs for core metabolic functions and environmental chemical metabolism. We identified long chain alkane utilization in two of the urine MAGs. Long chain alkanes are common pollutants that result from industrial combustion processes and end up in urine. This is the first study, to our knowledge, to demonstrate environmental chemical degradation potential in urine microbes through genome-resolved metagenomics. These findings lay the foundation for future evaluation of urobiome function in relation to health and disease.
Virginia Rich (Committee Member)
Vanessa Hale (Advisor)
Brian Husbands (Committee Member)
118 p.
Biomedical Research; Microbiology; Molecular Biology
urobiome, low biomass, genome-resolved metagenomics, host depletion, DNA extraction, microbiome, urine, canine

Recommended Citations

Citations

  • Lewis, Z. J. (2024). Urobiome: State of Knowledge, and Evaluation of Methods for Enabling Genome-Resolved Metagenomics of Urinary Tract Microbiota [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1720651049588034

    APA Style (7th edition)

  • Lewis, Zachary. Urobiome: State of Knowledge, and Evaluation of Methods for Enabling Genome-Resolved Metagenomics of Urinary Tract Microbiota. 2024. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1720651049588034.

    MLA Style (8th edition)

  • Lewis, Zachary. "Urobiome: State of Knowledge, and Evaluation of Methods for Enabling Genome-Resolved Metagenomics of Urinary Tract Microbiota." Master's thesis, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1720651049588034

    Chicago Manual of Style (17th edition)

osu1720651049588034
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This open access ETD is published by The Ohio State University and OhioLINK.