The human urinary microbiome; bacterial DNA in voided urine of asymptomatic adults

Abstract

The urinary microbiome of healthy individuals and the way it alters with ageing have not been characterized and may influence disease processes. Conventional microbiological methods have limited scope to capture the full spectrum of urinary bacterial species. We studied the urinary microbiota from a population of healthy individuals, ranging from 26 to 90 years of age, by amplification of the 16S rRNA gene, with resulting amplicons analyzed by 454 pyrosequencing. Mid-stream urine (MSU) was collected by the “clean-catch” method. Quantitative PCR of 16S rRNA genes in urine samples, allowed relative enumeration of the bacterial loads. Analysis of the samples indicates that females had a more heterogeneous mix of bacterial genera compared to the male samples and generally had representative members of the phyla Actinobacteria and Bacteroidetes. Analysis of the data leads us to conclude that a “core” urinary microbiome could potentially exist, when samples are grouped by age with fluctuation in abundance between age groups. The study also revealed age-specific genera Jonquetella, Parvimonas, Proteiniphilum, and Saccharofermentans. In conclusion, conventional microbiological methods are inadequate to fully identify around two-thirds of the bacteria identified in this study. Whilst this proof-of-principle study has limitations due to the sample size, the discoveries evident in this sample data are strongly suggestive that a larger study on the urinary microbiome should be encouraged and that the identification of specific genera at particular ages may be relevant to pathogenesis of clinical conditions.

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

“…Not only does it minimize protein degradation due to protease activity, [24][25][26] it also limits metabolite changes that can occur due to bacterial growth at RT, 27 and potential microbial growth due to the nonsterile nature of urine. 28 Glycine could be unambiguously identified and was found to be poorly reproducible.…”

“…The pellet can be used for DNA-based analyses such as genotyping 35 or urinary metagenomics analyses. 28 Microparticles in the supernatant can be isolated for further analyses by ultracentrifugation at a later stage, after storage at -80°C. 21,36 Urine preanalytical conditions have been studied in specific proteomic methods including MALDI-TOF-MS, 37 2D-electrophoresis, 4 SELDITOF MS, 31 and LC-MS, 38 showing that collection and processing variables can have a major impact on specific proteins and peptides.…”

Metabolic, Immune, and Gut Microbial Signals Mount a Systems Response to Leishmania major Infection

“…Presence of microbial genes was confirmed via 35 cycles of PCR [30 sec at 95°C, 40 sec at 55°C, 60 sec at 72°C], using primers against V1-V3 region of bacterial 16S rRNA genes as described by Lewis et al, 2013. 25 DNA samples were sequenced via next-generation pyrosequencing using a 454 FLX Genome Sequencer (Roche) by…”

Urine Is Not Sterile: Use of Enhanced Urine Culture Techniques To Detect Resident Bacterial Flora in the Adult Female Bladder

“…Yet, emerging evidence indicates that the lower urinary tract can have a urinary microbiota (3)(4)(5)(6)(7)(8). For example, our group previously reported the use of 16S rRNA gene sequencing to identify bacterial DNA (urinary microbiome) in culture-negative urine specimens collected from women diagnosed with pelvic prolapse and/or urinary incontinence, as well as from urine of women without urinary symptoms (4).…”