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Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
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Quantitative microbiome profiling links gut community variation to microbial load.

Doris Vandeputte1,2,3, Gunter Kathagen1,2, Kevin D'hoe1,2,3

  • 1KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute, Herestraat 49, B-3000 Leuven, Belgium.

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Quantitative microbiome profiling, using counts instead of ratios, reveals microbial load variations and their impact on host health. This method overcomes limitations of relative profiling, offering deeper insights into gut microbiota interactions and disease associations.

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Area of Science:

  • Microbiome research
  • Gut microbiota
  • Host-microbe interactions

Background:

  • Current faecal microbiota analyses use relative profiling, limiting understanding of host-microbe interplay.
  • Relative approaches cannot quantify microbial load or link it to host physiological data.
  • Altered microbial abundance itself may indicate disease-associated ecosystem changes.

Purpose of the Study:

  • To develop a workflow for quantitative microbiome profiling of faecal material.
  • To enable genuine characterization of host-microbiota interactions by using microbial counts.
  • To investigate the impact of microbial load on host health and disease.

Main Methods:

  • Parallelization of amplicon sequencing and flow cytometric enumeration of microbial cells.
  • Quantitative microbiome profiling of faecal samples.
  • Comparative analysis of relative vs. quantitative microbiome data.

Main Results:

  • Observed up to tenfold differences in microbial loads among healthy individuals, linked to enterotype differentiation.
  • Demonstrated that microbial abundances explain microbiota variation and host phenotype covariation.
  • Identified microbial load as a key driver of microbiota alterations in Crohn's disease patients.

Conclusions:

  • Quantitative microbiome profiling is essential for understanding host-microbe interactions beyond relative abundance.
  • Microbial load, not just relative abundance, is crucial for characterizing gut ecosystems and health.
  • This approach overcomes limitations of relative profiling, revealing true microbial dynamics and disease associations.