How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches

Frontiers in Cellular and Infection Microbiology, vol. 10, pp. 403

Abstract

Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g., flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extent different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: (1) microbial cell counting using flow cytometry (QMP), (2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and (3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.

Authors 15

1. Gianluca Galazzo

   Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute

   Affiliation as printed

   Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

2. Niels van Best Aachen Institute of Medical Microbiology

   Maastricht University Medical Centre · Maastricht University · Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

   Institute of Medical Microbiology, RWTH University Hospital Aachen, Aachen, Germany

3. Birke J. Benedikter

   Philipps University of Marburg · Maastricht University Medical Centre · Maastricht University · German Center for Lung Research · Universities of Giessen and Marburg Lung Center

   Affiliation as printed

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

   Department of Respiratory Medicine, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

   Member of the German Center for Lung Research (DZL), Universities of Giessen and Marburg Lung Centre, Institute for Lung Research, Philipps-University Marburg, Marburg, Germany

4. Kevin J. H. Janssen

   Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute

   Affiliation as printed

   Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

5. Liene Bervoets

   Maastricht University Medical Centre · Maastricht University

   Affiliation as printed

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

6. Christel Driessen

   Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute

   Affiliation as printed

   Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

7. Melissa Oomen

   Maastricht University Medical Centre · Maastricht University

   Affiliation as printed

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

8. Mayk Lucchesi

   Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute

   Affiliation as printed

   Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

9. Pascalle H. van Eijck

   Maastricht University Medical Centre · Maastricht University

   Affiliation as printed

   Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

10. Heike E. F. Becker

    Maastricht University Medical Centre · Maastricht University

    Affiliation as printed

    Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

    Division of Gastroenterology-Hepatology, Department of Internal Medicine, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

11. Mathias Walter Hornef Aachen Institute of Medical Microbiology

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Institute of Medical Microbiology, RWTH University Hospital Aachen, Aachen, Germany

12. Paul H. M. Savelkoul

    Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute · Amsterdam UMC Location Vrije Universiteit Amsterdam

    Affiliation as printed

    Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, Netherlands

    Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

    Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

13. Frank R. M. Stassen

    Maastricht University Medical Centre · Maastricht University

    Affiliation as printed

    Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands

14. Petra F. G. Wolffs

    Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute

    Affiliation as printed

    Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

15. John Penders corresponding

    Maastricht University Medical Centre · Maastricht University · Care and Public Health Research Institute

    Affiliation as printed

    Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, Netherlands

    Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands