Determination of the Optimal Bacterial DNA Extraction Method to Explore the Urinary Microbiota

Abstract

Recent advances in molecular biology have been successfully applied to the exploration of microbiota from various fluids. However, the urinary microbiota remains poorly explored, as its analysis requires specific technical considerations. Indeed, urine is a low microbial biomass environment, in which the representativity of each bacterium must be respected to obtain accurate data. Thus, sensitive extraction methods must be used to obtain good quality DNA while preserving the proportions between species. To address this, we compared the efficiency of five extraction methods on artificial urine samples spiked with low amounts of four bacteria species. The quality of the DNA obtained was further evaluated by different molecular biology approaches, including quantitative PCR and amplicon-based next-generation sequencing (NGS). Although two extraction methods allowed DNA of sufficient quality for NGS analysis to be obtained, one kit extracted a larger amount of DNA, which is more suitable for the detection of low-abundant bacteria. Results from the subsequent assessment of this kit on 29 human clinical samples correlated well with results obtained using conventional bacterial urine culture. We hope that our work will make investigators aware of the importance of challenging and adapting their practice in terms of the molecular biology approaches used for the exploration of microbiota.

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

“…Hence, sufficient DNA was extracted from the spiked samples for the sequencing steps within 35 minutes. Previous studies have also reported that the Blood and Tissue kit is best for DNA isolation from artificial human urine samples and canine urine (41,42). Karstens et al (43) also reported higher DNA yield from human urine by the Blood and Tissue kit, although DNA produced by both BiOstic and Blood and Tissue kits performed well in downstream analysis.…”

“…One of the critical steps for metagenomics-based diagnostic methods is DNA extraction, as variation in the extraction protocol can severely affect sequencing results. While studies have been carried out to determine the extraction efficiencies of different extraction kits on samples such as stool and blood, the urinary microbiome is relatively less explored (21,22). Some of the challenges encountered when working with urine samples are limited microbial biomass compared to other samples, the presence of different salt and urine components that can interfere with the extraction, and the presence of different assay inhibitors (23).…”

“…The QIAamp BiOstic Bacteremia DNA Kit, DNeasy Blood & Tissue Kit, and PowerFood Microbial Kit have been the most used kits for the isolation of DNA from urine samples. Particularly, the BiOstic Bacteremia DNA Kit and the Blood & Tissue Kit were found to preserve the bacterial diversity of the sample while yielding a large amount of good-quality DNA for downstream sequencing (21,24,25).…”

Urinary Microbiome of Reproductive-Age Asymptomatic European Women

“…PBS was discarded by centrifugation at 10,500 rpm/15 min/4°C immediately before genomic extraction. Genomic DNA from urine samples was extracted using the Qiagen DNeasy blood and tissue kit (Qiagen, Germany) according to the manufacturer’s protocol, using pretreatment for Gram-negative bacteria ( 51 ). DNA was eluted into 50 μL of Tris-HCl (pH 8.0) and stored at 4°C.…”

Synthesis of current pediatric urinary microbiome research

“…Additionally, it is important to critically evaluate and identify gaps in current benchmarking and optimization studies for pediatric urobiome applications. For example, many of the laboratory methods have been evaluated for optimization on adult human and canine urines ( 88 – 90 ), and several comparisons of detection methods ( 91 ), and bioinformatics ( 38 , 41 ) specifically for the urobiome. It is also essential that investigators in the pediatric urobiome research community employ current recommendations developed by the urobiome ( 15 ), kidney microbiome ( 92 ), and general microbiome communities ( 93 ) and develop consensus on standards and guidelines for the field.…”