The urinary microbiota composition remains stable over time and under various storage conditions

Background New sensitive techniques have revealed a large population of bacteria in the human urinary tract, challenging the perception of the urine of healthy humans being sterile. While the role of this urinary microbiota is unknown, dysbiosis has been linked to disorders like urgency urinary incontinence and interstitial cystitis. When comparing studies it is crucial to account for possible confounders introduced due to methodological differences. Here we investigated whether storage condition or time of collection, had any impact on the urinary microbial composition. Results For comparison of different storage conditions, urine was collected from five healthy adult female donors, and analyzed by 16S rRNA gene sequencing. Using the same methods, the daily or day-to-day variation in urinary microbiota was investigated in nineteen healthy donors, including four women, five men, five girls, and five boys. With the exception of 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 the urinary microbiota was found to be highly resilient to changes introduced by storage temperature and duration. In addition, we did not observe any intrapersonal daily or day-to-day variations in microbiota composition in women, girls or boys. Together our study supports flexibility in study design, when conducting urinary microbiota studies. Author summary The discovery of bacteria native to the urinary tract in healthy people, a location previously believed to be sterile, has prompted research into the clinical potential of these bacteria. However, methodological weaknesses can significantly influence such studies, and thus development of robust techniques for investigating these bacteria are needed. In the present study, we investigated whether differences in storage following collection, could affect the bacterial composition of urine samples. Next, we investigated if this composition exhibited daily or day-to-day variations. Firstly, we found, that the bacterial composition of urine could be maintained by storage at −80 °C, −20 °C, or refrigerated at 4 °C. Secondly, the bacterial composition of urine remained stable over time. Overall, the results of this study provide information important to study design in future investigations into the clinical implications of urinary bacteria.


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Abstract: 25 Background: New sensitive techniques have revealed a large population of bacteria in the human urinary tract, 26 challenging the perception of the urine of healthy humans being sterile. While the role of this urinary microbiota 27 is unknown, dysbiosis has been linked to disorders like urgency urinary incontinence and interstitial cystitis.

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When comparing studies it is crucial to account for possible confounders introduced due to methodological 29 differences. Here we investigated whether storage condition or time of collection, had any impact on the urinary 30 microbial composition.

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Results: For comparison of different storage conditions, urine was collected from five healthy adult female 32 donors, and analyzed by 16S rRNA gene sequencing. Using the same methods, the daily or day-to-day variation 33 in urinary microbiota was investigated in nineteen healthy donors, including four women, five men, five girls, 34 and five boys. With the exception of two male adult donors, none of the tested conditions gave rise to significant 35 differences in alpha and beta diversities between individuals. Conclusion: The composition of the urinary 36 microbiota was found to be highly resilient to changes introduced by storage temperature and duration. In 37 addition, we did not observe any intrapersonal daily or day-to-day variations in microbiota composition in 38 women, girls or boys.

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Together our study supports flexibility in study design, when conducting urinary microbiota studies.

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Author summary 42 The discovery of bacteria native to the urinary tract in healthy people, a location previously believed to be sterile, 43 has prompted research into the clinical potential of these bacteria. However, methodological weaknesses can 44 significantly influence such studies, and thus development of robust techniques for investigating these bacteria day-to-day variations.  Staphylococcus,and Prevotella[13,[18][19][20][21][22][23]. It is assumed that these bacteria provide healthy 6 115 Urine microbiota composition is independent of daily and day-to-day variation 116 First morning urine is often more concentrated than subsequent urine samples throughout the day, while 117 differences in daily routines (e.g. sleep rhythm, diet, sexual activity or exercise) may introduce variations during 118 the day. We therefore speculated that morning urine could contain higher bacterial loads, and possibly a 119 different bacterial composition than urine collected in the evening. To test this hypothesis, we compared urine 120 samples collected in the morning and evening on two independent days from 19 healthy donors (4 women, 5 121 men, 5 girls, and 5 boys). Following DNA extraction, the resulting DNA yield ranged from <2 to 218.25 ng per mL 122 urine. Importantly, DNA yield did not differ based on within day or day-to-day (data not shown).  195 In conclusion, we showed that the urinary microbiota is stable over time, and that sub-optimal temperatures for 196 urine storage may be used. We recommend, however, that samples be transferred to -80 °C as quickly as possible 197 after collection, to avoid loss of the already limited DNA in urine samples. In addition, we highly recommend that

Study participants and urine collection 207
In total, 25 healthy volunteers, without symptoms from the bladder (based on self-reporting in a questionnaire 208 prior to study participation) or intake of any antibiotics within the past 3 months, were included into this study.

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Furthermore, in cases where medicine or hormonal contraceptives were used, these should be taken within the 210 same period on all study days. Use of non-prescription painkillers was not accepted for up to 24 hours prior to 211 urine collection. In addition, the study participants were instructed to avoid urine collection during menstruation, 212 and pregnant women were not included into the study. The identity of all donors was anonymous and no 213 personal data was registered, besides the sex and age interval of which they belonged to. For the initial study on 214 different storage conditions, 5 women were recruited, and for the following study on daily or day-to-day 215 variations, 20 participants were recruited encompassing 5 men (18-50 years), 5 women (18-50 years), 5 boys (5-216 10 years), and 5 girls (5-10 years). One woman was however excluded from the latter of the two studies, due to 217 incorrect storage of the collected urine sample, leaving 19 participants in total.

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For the study on storage conditions, urine was collected at the laboratory by the clean catch method. Samples 219 were immediately aliquoted in tubes with 10 mL urine and transferred to the specified storage conditions as 220 summarized in Fig 1,  storage conditions, the following primer sequences were utilized (Forward: 5'-GTGCCAGCMGCCGCGGTAA-3',   253  reverse: GGACTACHVGGGTWTCTAAT), while for the study investigating variations between evening and morning   254 and day-to-day, a slightly modified reverse primer was used (5'-GGACTACNVGGGTWTCTAAT-3'). Samples were 255 pooled and sequencing was performed on a MISeq (Illumina, USA), as previously described [38]. To measure error 256 rate during sequencing and batch effects, a 20 % PhiX control library was added. As negative control, nuclease-257 free water was used, while a complex sample obtained from an anaerobic digester system was utilized as a 258 positive control.