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Species-level urinary microbiome composition in pediatric population
Lisa Karstens, PhD1, Tatyana Sysoeva, PhD2, Maryellen Kelly, DNP, CPNP, MHSc3.
1Oregon Health Sciences University, Portland, OR, USA, 2University of Alabama, Huntsville, Huntsville, AL, USA, 3Duke University, Durham, NC, USA.

Background: Studies in adults report associations between recurrent urinary tract infections (UTI) and the composition of the urinary microbiome (urobiome). There is a lack of knowledge surrounding the composition and development of the urobiome in children. We tested the feasibility of a novel sequencing approach to establish the species-level composition of the urobiome of children.
Methods: We obtained catheterized urine samples from fifty children ages 0-60 months who underwent voiding cystourethrograms at a single institution. Variables collected were sex, age, UTI history, antibiotic use history, and current antibiotic use. Urine biomass was collected via vacuum filtration of urine on 0.22μm filters and total DNA was extracted using an optimized version of QIAamp Fast DNA Stool Mini Kit with included lysozyme. 16S rRNA gene sequencing using full-length synthetic long-read technology was performed in triplicate along with positive and negative controls. Bacterial sequences were processed with Dada2 and taxonomy was assigned using the Silva database. The alpha diversity measures were compared between groups using non-parametric Wilcoxon rank-sum.
Results: Preliminary analysis shows that bacterial DNA can be robustly detected in young children of both sexes as early as 3 months, with 33 of 50 (66%) urine samples providing analyzable sequencing data ("sequence positive"). Technical replicates demonstrated consistency in the relative abundance of bacteria, indicating that the data obtained is robust. No significant associations between demographics or clinical variables from individuals whose samples were sequence positive versus sequence negative were identified. There were significant differences by sex in alpha diversity assessed by the Inverse Simpson and Shannon indices (p = 0.05, 0.04 respectively, Figure A). Overall, age was significantly associated with alpha diversity as measured by the observed number of species (p = 0.007) and Shannon index (p = 0.02). When investigating each sex independently, males demonstrated significant associations with age in terms of the observed number of species (p = 0.02). Both males and females have a polymicrobial urobiome composed of several bacteria (Figure B).
Conclusion: This is the first species-level analysis of the urobiome in the pediatric population. This preliminary study demonstrates that the pediatric microbiome can be characterized using this approach by combining filtering and synthetic long-read sequencing. It also suggests that differences in the urobiome exists between sexes at an early age.

Figure A. Alpha diversity measured by the Inverse Simpson and Shannon diversity indices are significantly different between males and female children (Wilcoxon rank-sum p = 0.05 and 0.04, respectively).

Figure B. Stacked bar plot providing an overview of the urobiome composition of female and male children.

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