Questions and challenges associated with studying the microbiome of the urinary tract

Abstract

Urologists are typically faced with clinical situations for which the microbiome may have been a contributing factor. Clinicians have a good understanding regarding the role of bacteria related to issues such as antibiotic resistance; however, they generally have a limited grasp of how the microbiome may relate to urological issues. The largest part of the human microbiome is situated in the gastrointestinal tract, and though this is mostly separated from the urinary system, bacterial dissemination and metabolic output by this community is thought to have a significant influence on urological conditions. Sites within the urogenital system that were once considered "sterile" may regularly have bacterial populations present. The health implications potentially extend all the way to the kidneys. This could affect urinary tract infections, bladder cancer, urinary incontinence and related conditions including the formation of kidney stones. Given the sensitivity of the methodologies employed, and the large potential for contamination when working with low abundance microbiomes, meticulous care in the analyses of urological samples at various sites is required. This review highlights the opportunities for urinary microbiome investigations and our experience in working with these low abundance samples in the urinary tract.

Keywords: Microbiome; low bacterial abundance; sample contamination.

Conflict of interest statement

Conflicts of Interest: The authors have no conflicts of interest to declare.

Figures

Figure 1

Introduction of non-target DNA and other impairments of next generation sequencing microbiome analysis in the urinary tract. 1. Sample collection. Bacteria occur in different densities with different host materials such as tissue, urine or kidney stones. (a) The bacterial composition in urine is altered by capture fraction and represent different parts of the urinary tract, it is also effected by patient sex and age; (b) Entry to the upper urinary tract via (a) open surgical procedures or even by (b) minimally invasive technologies still risks bacterial acquisition via the skin and top layers of dermal tissue. 2. Tissue processing and DNA extraction. (a) Samples of renal tissue that may contain small amounts of bacteria will rapidly bind up the column with DNA from the host. To prevent microbial DNA loss, alternatives to column capture is required in low abundance microbiome in tissue samples. Free DNA and DNA in dead bacteria on the surface can be made unavailable by PMA treatment before tissue homogenization; (b) kidney stones will have some human DNA and the surface can be sterilized before pulverization in a sterile bag; (c) bacteria in urine are typically more abundant than samples from other parts of the urinary tract. Optimal processing requires greater than 10 mL in our experience and bacteria should be stored separated by pelleting of the sample by centrifugation; (d) DNA extraction kits and their reagents contain trace amounts of bacterial DNA, not normally a problem in high abundance samples, but potentially a menace for the urinary tract; (e) The amount of contaminating background DNA is different depending upon the origin of the material and abundance of commensal bacteria. 3. PCR amplification. PMA treatment of PCR reagents will reduce some of the background DNA contamination, but (a) aerosols and cross contamination from other samples is likely to occur at some level. (b) Low abundance bacterial DNA and high background of other DNA, such as the host may allow non targeted amplification to occur, an example with some primer types is closely-related mitochondrial DNA. 4. Sequencing and analysis. High and low DNA concentration samples should not be combined together (e.g., urinary and faecal) because insufficient read depth will occur in the lower abundance samples. Numbers of sample per sequencing run should be normalized to expected diversity. Appropriate removal of contaminating sequences should be allowed for prior to undertaking further statistical analysis.