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

Julie A Vendrell,Elise Heckendorn,Sylvain Godreuil,Jérôme Solassol,Simon Cabello-Aguilar,Steven Henry

doi:10.3390/ijms23031336

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.

Highlights

Until the recent discovery of a unique urinary microbiota in the urinary tract [1,2,3], urine was assumed to be a sterile environment in healthy people
To select the best extraction method for analysis of the microbiome of urine samples, we used a large volume of sterilized urine that we spiked with four species of bacteria at different abundances: Lactobacillus delbrueckii and Enterococcus faecalis were spiked at a high abundance, Prevotella bivia and Escherichia coli at a low abundance
Despite the latest advances in molecular biology, the urinary microbiota still remains a poorly explored field of investigation, as working with this type of fluid is quite challenging for various reasons: (i) urine is composed of a very low microbial biomass compared with other types of samples; (ii) samples have different salt concentrations that may disturb

Summary

Introduction

Until the recent discovery of a unique urinary microbiota in the urinary tract [1,2,3], urine was assumed to be a sterile environment in healthy people. Some bacteria from the urinary commensal microbiota have recently been reported to promote/cause infections [4], urinary incontinence [5,6], kidney transplant rejection or dysfunction [6,7], and cancer development [8,9,10]. Standard urine cultures have long been used in clinical microbiology laboratories to detect uropathogens, especially during a urinary tract infection (UTI) episode. These analyses, which allow the detection of viable bacteria, exhibit several important limitations: they are less adapted to the detection of anaerobic and slowly growing microorganisms and do not allow the detection of unknown pathogens. There have been recent improvements to urine culture techniques, modification of culture media and identification of isolated species by matrix-assisted laser desorption ionization–time of flight mass spectrometry [5], these semiquantitative techniques are not fully adapted for

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Conclusion