Establishing and analyzing the Simplified Human Intestinal Microbiota (SIHUMI) as a versatile in vitro gut microbiome model with qPCR-based strain-level tracking.

Abstract

A major challenge in microbiome research is the inherent complexity and inter-individual variability of the human gut microbiota. To address this, we have developed a detailed protocol for establishing and analyzing a Simplified Human Intestinal Microbiota (SIHUMI)-a defined, in vitro bacterial consortium composed of seven fully sequenced and anaerobically culturable human gut commensals. This model enables highly reproducible and controlled experiments, in which the individual growth of each member can be quantitatively tracked over time (up to 48 h) via species-specific qPCR. The protocol outlines optimized and standardized steps, including consortium setup, time-resolved sample collection, DNA extraction and qPCR analysis. It can be used to evaluate community dynamics in response to interventions such as nutrients, antimicrobials or other xenobiotics. The system is readily adaptable: additional strains can be incorporated, including pathogens (e.g., Clostridioides difficile), to transform it into an infectious disease model. In addition, we describe two optional rapid methods for assessing interspecies interactions and provide an open-source web app for generating interaction network plots. This enables exploration of ecological mechanisms and potential off-target effects. The entire workflow-from setup to data acquisition-can be completed within 1 week. This qPCR-based protocol offers a validated and accessible platform for gut microbiome research, providing a standardized, strain-level and time-resolved alternative to 16S- or fluorescence-based workflows and enabling quantitative, scalable analysis of defined microbial communities.