Metaproteomics Reveals Growth Phase-Dependent Responses of an in Vitro Gut Microbiota to Metformin

Metaproteomics has been used in combination with in vitro gut microbiota models to study drug-microbiome interactions. However, it remains unexplored whether the metaproteomics profile of in vitro gut microbiota responds differently to a same stimulus added at different growth phases. In this study, we cultured a human gut microbiota in 96-deep well plates using a previously validated model. Metformin was added during the lag, log, and stationary phases. Microbiome samples, collected at different time points, were analyzed by optical density and function by metaproteomic. The in vitro gut microbiota growth curves, taxonomy, and functional responses were different depending whether metformin was added during the lag, log, or stationary phases. The addition of drugs at the log phase may lead to the greatest decline of bacterial growth. Metaproteomic analysis suggests that the strength of the metformin effect on the gut microbiome functional profile may be ranked as lag phase > log phase > stationary phase. Metformin added at the lag phase may result in a significantly reduced level of the Clostridiales order and an increased level of the Bacteroides genus, which is different from stimulations during the rest of the growth phases. Metformin may also result in alterations of several pathways, including energy production and conversion, lipid transport and metabolism, translation, ribosomal structure, and biogenesis. Our results indicate that the timing for drug stimulation should be considered when studying drug-microbiome interactions in vitro.