Voided Urinary Microbiota Is Stable Over Time but Impacted by Post Void Storage.
Author(s): Bundgaard-Nielsen C, Ammitzbøll N, Isse YA, Muqtar A, Jensen AM, Leutscher PDC, Arenholt LTS, Hagstrøm S, Sørensen S
Publication: Front Cell Infect Microbiol, 2020, Vol. 10, Page 435
PubMed ID: 32984068 PubMed Review Paper? No
Purpose of Paper
This paper compared DNA yields and urinary microbial DNA composition among urine specimens from healthy women that were processed immediately or stored at 4°C, -20°C and -80°C before DNA extraction and among urine collected from men, women, boys and girls in the evening as opposed to the morning and on a weekday versus a weekend.
Conclusion of Paper
In the majority of specimens, the highest yield was obtained when DNA was extracted from fresh urine followed by urine stored at -80°C; significantly lower levels of DNA were obtained from some specimens stored at 4°C for ≥4 h or -20°C for ≥24 h compared to those processed immediately. Alpha diversity was comparable when urine specimens were processed immediately or stored at -80°C, but significant differences that were dependent on storage duration were noted for select specimens following storage at 4°C and -20°C. In contrast, significant differences in Shannon diversity were limited to a decrease in one specimen following storage at -20°C for 72 h. Importantly, operational taxonomic units (OTUs) abundances clustered specimens by patient rather than storage temperature/duration.
The authors report that DNA yield did not vary between days (weekday versus weekend) or time of day (morning versus evening) of collection. Although, some variability was found in alpha diversity between morning and evening urine specimens and specimens collected on the weekend versus weekday, the differences were small and the direction of change was inconsistent. Further, beta-diversity showed that the microbial composition was stable among time-points for all volunteers, with the exception of specimens from two men. Most specimens clustered by a patient’s gender and age, but a single specimen from a man clustered with those from women.
Studies
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Study Purpose
This study compared DNA yields and urinary microbial DNA composition among urine specimens from healthy women that were processed immediately and those that were stored at 4°C, -20°C and -80°C before DNA extraction. Clean catch midstream urine was collected from five healthy females into special cups. Aliquots were immediately processed or stored in duplicate at 4°C for 4, 24, and 72 h; at -20°C for 24 and 72 h; and at -80°C for >72 h before processing. Urine was centrifuged at 3000 x g for 20 min, the pellet was resuspended in lysis buffer and lysed with a TissueLyser LT instrument. Bacterial DNA was isolated using the QIAamp Viral RNA Mini Kit. DNA yield was quantified using a NanoDrop machine and a Qubit dsDNA HS Assay Kit. 16S rRNA libraries were constructed with the DNAsense Kit and sequenced on a MiSeq instrument. Data was analyzed following the UPARSE workflow.
Summary of Findings:
In four of the five specimens evaluated, the highest yield was obtained when DNA was extracted from fresh urine followed by urine stored at -80°C. In three of the five specimens, significantly lower levels of DNA were obtained when urine specimens were stored at 4°C for 4 h or at -20°C for 72 h than when processed immediately (P<0.05 for all). Significant declines relative to immediately processed specimens were also evident in two specimens following storage at 4°C for ≥24 h (P<0.05, all) and in one specimen following storage at -20°C for 24 h (P<0.05). Using 16S rRNA sequencing, a total of 933 operational taxonomic units (OTUs) were identified, of which 96.1% were identified at the phylum level and 53.2% of which were identified at the genus level. Alpha diversity was comparable when specimens were processed immediately or stored at -80°C, but significant differences were observed in some but not all specimens following storage at 4°C and -20°C depending on storage duration. In contrast, significant differences in Shannon diversity were limited to a decrease in one specimen following storage at -20°C for 72 h relative to immediately processed urine specimens. Importantly, OTU abundances clustered specimens by patient rather than storage temperature/duration. Further examination revealed an increase in bacteria of the genus Gardnerella in urine from one patient and Dialister in urine from another following storage at 4°C.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry Cell count/volume Next generation sequencing DNA Next generation sequencing DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 h
4 h
24 h
72 h
>72 h
Storage Storage temperature 4℃
-20℃
-80℃
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Study Purpose
This study compared DNA yields and urinary microbial DNA composition among urine specimens collected at different times of day and on different days from healthy women, men, girls and boys. Midstream urine was self-collected in the morning and evening of a weekday and a weekend day from 19 healthy volunteers: 4 women (age 18-50 years), 5 men (age 18-50 years), 5 girls (age 5-10 years), and 5 boys (age 5-10 years). After collection, volunteers were instructed to freeze the urine at home and deliver it to the laboratory within 24 h. Urine was stored at the laboratory at -80°C until DNA extraction. Urine was centrifuged at 3000 x g for 20 min, the pellet was resuspended in lysis buffer, and lysed with a TissueLyser LT instrument. Bacterial DNA was isolated using the QIAamp Viral RNA Mini Kit. DNA yield was quantified using a NanoDrop machine and the Qubit dsDNA HS Assay Kit. 16S rRNA libraries were constructed with the DNAsense Kit and sequenced on a MiSeq instrument. The 54 specimens that yielded less DNA than the negative control during library preparation PCR amplification were excluded from further analysis. Data was analyzed following the UPARSE workflow.
Summary of Findings:
The authors report that DNA yield did not vary between days (weekday versus weekend) or time of day (morning versus evening) of collection. Interestingly, none of the specimens that yielded less DNA than the negative control during library preparation PCR amplification were from women, but 16 (40%) were from girls, 26 (65%) were from men, and 12 (30%) were from boys. The remaining specimens had good coverage and resulted in a total of 2,538 OTUs, of which 93.1% were identified at the phylum level and 50.9% of which were identified at the genus level. Some variability was observed in alpha diversity between morning and evening specimens and specimens collected on different days, but the differences were small and the direction of change was inconsistent. Further, beta-diversity showed that the microbial composition was stable among time-points for all volunteers, except for those from two men. Most specimens clustered by a patient’s gender and age, but a single specimen from a man clustered with those from women.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry Cell count/volume Next generation sequencing DNA Next generation sequencing DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Biospecimen Acquisition Time of biospecimen collection Weekday
Weekend day
Morning
Evening
Preaquisition Patient age 18-50 years
5-10 years