A flexible high-throughput cultivation protocol to assess the response of individuals’ gut microbiota to diet-, drug-, and host-related factors

Abstract

SUMMARYAnaerobic cultivation of fecal microbiota is a promising approach to investigate how gut microbial communities respond to specific intestinal conditions and perturbations. Here, we describe a practical protocol using 96-deepwell plates to cultivate stool-derived gut microbiota. Our protocol addresses key challenges in high-throughput culturing, including a thorough assessment of the impact of gas phase on medium chemistry, a modular medium preparation process to enable testing of several conditions in parallel, a medium formulation designed to maximize the compositional similarity of fecal cultures with the donor microbiota, and the creation of a step-by-step protocol detailing all practical procedures from material preparation to sample handling for analyses. Finally, we validated the protocol by demonstrating that cultivated fecal microbiota responded similarly to dietary fibers (resistant dextrin, soluble starch) and drugs (ciprofloxacin, 5-fluorouracil) as reported in vivo. This high-throughput cultivation protocol can facilitate culture-dependent studies, accelerate the discovery of gut microbiota-diet-drug-host interactions, and pave the way to personalized and microbiota-centered interventions.MOTIVATIONThe human gut microbiota is a complex ecosystem unique to each individual. The extent of this diversity has limited our capacity to fully comprehend microbial dynamics that apply to the entire human population. To probe the response of donor-specific microbial communities to intestinal conditions or perturbations,in vitrocultivation of stool-derived gut microbiota can be employed. However, cultivating gut microbiota under strictly anaerobic conditions is commonly performed using individual gas-tight tubes, which is a highly time-consuming strategy that limits the number of conditions and donor microbiota that can be tested in parallel. A flexible high-throughput protocol to cultivate and test donor-specific gut microbiota is therefore required. Hence, we developed a robust procedure for cultivating stool-derived microbiota (and pure gut microbial cultures) in 96-deepwell plates within an anaerobic chamber.