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Sample Preparation Protocols for Urinary Microbiome Sequencing

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The urinary tract involves the kidney, bladder, ureter, urethra, and the urogenital tract that includes reproductive organs such as the vagina, periurethral skin, cervix, pubic skin surfaces, penis, and perineal area. This area of the body was described as sterile in the previous studies due to the physical and chemical properties of urine. But more recent research has proven that the urinary tract has its own microbiome, and its composition is different in male and female adults. The identified urinary microbiota may consist of species residing within the urinary tract, bladder, or urogenital tract depending on the sampling method used. As mentioned, the composition urinary microbiome may differ depending on age and sex. Recent studies revealed that bacteria belonging to the group called Corynebacterium are more likely to be found in males. This group of bacteria can also be found in human skin. While females are more likely to have Lactobacillus bacterium which is also found in the human gut.

Characterization of Urinary Microbiome

There have been several techniques developed to characterize the human urinary microbiome. Recent studies have shown that the human urinary microbiome biomass ranges from 100 to 105 colony-forming units per milliliter of urine. The three main sampling techniques for urine include (1) the collection of midstream clean-catch urine (CC), (2) the collection of urine through a transurethral catheter, and (3) the collection of urine via suprapubic aspiration. The mentioned sampling techniques have their own advantages and disadvantages, and most of these introduce perturbations to the local microbial communities. With that, sampling order is important to consider as it may result in the contamination of sequential sampling sites.

In order to characterize the entire microbial composition without culture bias, next-generation sequencing-based metagenomic sequencing approaches are applied. This has two major paths: (1) 16S rRNA amplicon sequencing, and (2) whole-genome shotgun metagenomic sequencing.

Acquisition and Preparation of Urine Sample

Prior to the urinalysis and urine culture, as well as 16S rRNA amplicon sequencing, urine samples are collected by midstream voided collection. This is then followed by sterile catheterization of the bladder as the individual could drink water and allow the bladder to refill naturally. The collected urine sample must be immediately placed at 4°C and must be processed within six hours for a standard urinalysis and urine culture.

The samples will be processed for sequencing through centrifugation at 4°C, 5,000 ×g, for 20 minutes. The resulting pellet must be recovered together with approximately one milliliter of residual urine supernatant to avoid disturbing the pellet. Ten-milliliter ice-cold phosphate-buffered saline must be added, followed -by gentle shaking of the tube. Another centrifugation process must be done at 4°C, 5,000 ×g, and this time for 15 minutes. The wet urinary pellet must be frozen at -80°C until the process of lysis was performed using two milliliters of 10mM ammonium bicarbonate buffer. This buffer contains 0.1% Triton-X100, 0.5% octylglucoside, 5-µg/mL leupeptin, 10mM ethylenediaminetetraacetic acid and 2mM 2,6-Dichlorobenzamide. The suspensions must be heated to 85°C for 5 minutes and sonicated at amplitude 4 (Misonex 3000 sonicator) in 30 seconds on/15 seconds off cycles 10 times on the ice. The suspension must be centrifugated again at 4°C, 16,100 ×g, for 15 minutes and the supernatant must be recovered. Lastly, the genomic DNA will be isolated from the pelleted sample.

References

  1. Neugent ML, Hulyalkar NV, Nguyen VH, et al. Advances in Understanding the Human Urinary Microbiome and Its Potential Role in Urinary Tract Infection. mBio. 2020, 11(2).
  2. Pohl HG, Groah SL, Pérez-Losada M, et al. The urine microbiome of healthy men and women differs by urine collection method. International Neurourology Journal. 2020, 24(1):41.
  3. Tang J. Microbiome in the urinary system—a review. AIMS microbiology. 2017;3(2):143.
* For Research Use Only. Not for use in diagnostic procedures or other clinical purposes.

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