Optimizing ex vivo culture conditions to study human gut microbiome

Abstract

The inter-individual variations of gut microbiome contribute to the different responses toward drug therapy among populations, developing a reliable ex vivo culture method for mixed bacteria is the urgent need for predicting personal reaction to drug therapy. Unfortunately, very few attentions have been paid to the bias that could be introduced during the culture process for mixed bacteria. Here we systemically evaluated the factors that may affect the outcomes of cultured bacteria from human feces. We demonstrated that inter-individual difference of host gut microbiome was the main factor affecting the outcomes of cultured bacteria, followed by the culture medium and time point. We further optimized a new medium termed GB based on our established multi-dimensional evaluation method, which could mimic the status of in situ host gut microbiome to the highest extent. Finally, we assessed the inter-individual metabolism by host gut microbiome from 10 donors on three frequently used clinical drugs (aspirin, levodopa and doxifluridine) based on the optimized GB medium. Our results revealed obvious variation in drug metabolism by microbiome from different donors, especially levodopa and doxifluridine. This work suggested the optimized culture medium had the potential for exploring the inter-individual impacts of host gut microbiome on drug metabolism.

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

“…In comparison, 6C contained the carbohydrates found in the Macfarlane medium (15% starch, 15% pectin, 15% xylan, 8% arabinogalactan, 8% guar gum, and 38% inulin) [ 59 ], a nutrient-rich medium that closely mimics the gut chyme of healthy adults [ 59 ], and is commonly used in continuous intestinal in vitro models [ 60 ]. Finally, we incorporated two conditions designed to replicate the nutritional composition of media previously used for gut microbiota culturing, namely Brain Heart Infusion (BHI) medium [ 20 , 61 ] and Gut Microbiota Medium (GMM) [ 20 , 35 , 61 ]. Since we showed that the bicarbonate concentration affects the pH within the chamber, and given that BHI and GMM inherently lack bicarbonate, we formulated BHI-like and GMM-like media to improve the comparability of results without introducing confounding factors related to pH.…”

“…S5B ). A previous study suggested that the ex vivo overgrowth of Enterobacteriaceae in the starch-containing medium, modified Gifu Anaerobic Medium, could be prevented by mixing it with the Bryant and Burkey medium [ 18 , 20 ]. The latter contains acetate, a known inhibitor of Enterobacteriaceae taxa, such as Escherichia coli [ 92 ].…”

“…Cultivation with a higher throughput is thus crucial to advance our understanding of gut microbiota-diet-drug–host interactions and facilitate the development of personalized microbiota-centered interventions [ 4 , 14 ]. Different high-throughput cultivation methods have been developed for multiwell plates in the anaerobic chamber [ 16–20 , 32 ], with important efforts directed toward evaluating and optimizing medium formulation [ 17 , 18 , 20 , 33–36 ], streamlining the workflow from feces to sample analysis [ 19 , 37 ], and providing detailed technical procedures for cultivation [ 38 ].…”

“…a taxonomic group associated with dysbiosis (e.g. irritable bowel syndrome, inflammatory bowel disease, obesity, colorectal cancer) [ 39 ] and prone to overgrowth during ex vivo cultivation [ 16 , 18 , 20 , 21 , 36 , 40 ]. At the core of our approach is the use of a standardized basal medium that can be flexibly modified to test fecal microbiota responses to factors related to diet, drugs, and the host, individually or in combination.…”

Advances in the isolation, cultivation, and identification of gut microbes

“…Overall, culturomics emphasizes the continuous refinement and enhancement of culture media and conditions to optimize species diversity through streamlined procedures [ 32 , 33 ]. One advantage of using solid media is its ability to facilitate visual identification and selection of microbiota based on their observable morphologies.…”