Background Recently, associations between recurrent urinary tract infections (UTI) and the urinary microbiome (urobiome) composition have been identified in adults. However, little is known about the urobiome in children. We aimed to characterize the urobiome of children with species-level resolution and to identify associations based on UTI history.

Study design Fifty-four children (31 females and 21 males) from 3 months to 5 years of age participated in the study. Catheterized urine specimens were obtained from children undergoing a clinically indicated voiding cystourethrogram. To improve the analysis of the pediatric urobiome, we used a novel protocol using filters to collect biomass from the urine coupled with synthetic long-read 16S rRNA gene sequencing to obtain culture-independent species-level resolution data. We tested for differences in microbial composition between sex and history of UTIs using non-parametric tests on individual bacteria and alpha diversity measures.

Results We detected bacteria in 61% of samples from 54 children (mean age 40.7 months, 57% females). Similar to adults, urobiomes were distinct across individuals and varied by sex. The urobiome of females showed higher diversity as measured by the inverse Simpson and Shannon indices but not the Pielou evenness index or number of observed species (p = 0.05, p=0.04, p = 0.35, and p = 0.11, respectively). Additionally, several species were significantly overrepresented in females compared to males, including those from the genera Anaerococcus, Prevotella, and Schaalia (p = 0.03, 0.04, and 0.02, respectively). Urobiome diversity increased with age, driven mainly by males. Comparison of children with a history of 1, 2, or 3+ UTIs revealed that urobiome diversity significantly decreases in the group that experienced 3+ UTIs as measured by the Simpson, Shannon, and Pielou indices (p = 0.03, p = 0.05, p = 0.01). Several bacteria were also found to be reduced in abundance.

Discussion In this study, we confirm that urobiome can be identified from catheter-collected urine specimens in infants as young as 3 months, providing further evidence that the pediatric bladder is not sterile. In addition to confirming variations in the urobiome related to sex, we identify age-related changes in children under 5 years of age, which conflicts with some prior research. We additionally identify associations with a history of UTIs.

Conclusions Our study provides additional evidence that the pediatric urobiome exists. The bacteria in the bladder of children appear to be affected by early urologic events and warrants future research.

The authors have declared no competing interest.

This work was funded by the National Institutes of Health (NIH) CAIRIBU U24 Interactions Core award U24-DK-127726 through the Collaboration Awards Program. LK was also supported by NIH NIDDK Award K01 DK116706. The contents of the article are solely our responsibility and do not represent the official views of the NIH or of any other funding agency.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The research study protocol was reviewed and approved by the Duke University Medical Center Institutional Review Board under protocol #Pro0010031. From 2018-2021 children from birth to age 5 years who had a voiding cystourethrogram (VCUG) conducted for a clinical indication were offered enrollment at a single institution under IRB #Pro0010031. At baseline, parents or guardians (hereafter parents) granted permission for a urine sample to be collected and their childs electronic medical record to be accessed. Parents provided written consent for their child to be included in the study. Identifiers presented in plots and data are deidentified and do not represent patient or research subject identifiers.

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Data produced in the present study are available upon reasonable request to the authors