Voided Urinary Microbiota Is Stable Over Time but Impacted by Post Void Storage

Abstract

Background: New sensitive techniques have revealed a thriving bacterial community in the human urinary tract, challenging the perception that urine in healthy humans is sterile. While the functional role of this urinary microbiota is unknown, dysbiosis has been linked to urgency urinary incontinence and risk of urinary tract infections. When comparing studies, it is crucial to account for possible confounders introduced due to methodological differences. Here we investigated whether collection and storage conditions had any impact on the urinary microbial composition. Results: For comparison of different storage conditions, midstream urine was collected from five healthy adult female donors and analyzed by 16S rRNA gene sequencing. Samples stored at −80 and −20 • C, but not 4 • C, were found to be comparable to freshly handled voided urine. Using the same methods, the daily or day-today variation in urinary microbiota was investigated in 19 healthy donors, including four women, five men, five girls, and five boys. Apart from two male adult donors, none of the tested conditions gave rise to significant differences in alpha and beta diversities between individuals. Conclusion: The composition of voided urinary microbiota was found to be effectively maintained by freezing, but not storage at 4 • C. In addition, we did not observe any intrapersonal daily or day-today variations in microbiota composition in women, girls or boys. Together our study supports present methodologies that can be used in future studies investigating the urinary microbiota.

Pre- and postmenopausal women have different core urinary microbiota

“…Fourth, we only collected urine at a single timepoint, and as the bacterial composition has been shown by some to fluctuate over time 33 , this possible lack of temporal stability may have impacted the results. We have, however, previously shown that the composition of the urinary microbiota remains stable over a shorter time period 42 . Finally, we did not account for other factors that could potentially influence the urinary microbiota, such as diet 43 , fluid intake, urinary oxygen concentrations 44 , menstruation cycle 33 , sexual activity 33 , and vulvo-vaginal symptoms 45 , 46 .…”

“…From 10 mL of urine, bacterial DNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen) according to manufacturer´s recommendations, modified with a pre-treatment step, as previously explained 42 . All samples were extracted and analyzed as duplicates.…”

Urinary microbiota; Which non‐ınvasive urine collection method should we use?

“…there is no complete consensus on the method of urine sample collection. [3][4][5][6][7][8][9] In comparison between the first and the midstream urine samples of our study, there is no statistically significant difference in almost all taxonomic levels. Only one statistically significant difference was found in the midstream urine contained higher levels of Clostridia in contrast to the first voided urine at the class and the order level comparisons.…”

“…2 Moreover there is no complete consensus on the urine sample collection method as many variations have been suggested in different studies. [3][4][5][6][7][8][9] Recently, it was shown that 16S ribosomal RNA sequencing can detect bacteria residing in the urinary tract from the urinary samples with a high sensitivity and specifity, even if the patient has UTI symptoms or not. 2,10 Therefore, we have decided to investigate the reliability of a two-step urine collection method and the validity of the hypothesis which argues that the first voided urine reflects the urethra and the midstream urine reflects the bladder.…”

Processing human urine and ureteral stents for 16S rRNA amplicon sequencing

“…Human urine and stent samples must be partially processed within 4–6 h of collection prior to their frozen storage in order to optimize downstream DNA yield ( Jung et al., 2019 , Bundgaard-Nielsen et al., 2020 ). Importantly, urine samples should not be refrigerated prior to their processing due to increased precipitation of urinary sediments, which has been shown to decrease DNA yield ( Ribeiro et al., 2013 , Ackerman et al., 2019 ).…”