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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 interaction. However, we don't know whether the metaproteomics profile of in vitro gut microbiota responds differently to a same stimuli 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, as the stimulus, was added at the lag, log and stationary phases of growth. Microbiome samples were collected at different time points for optical density and metaproteomic functional analysis. Results show that in vitro gut microbiota responded differently to metformin added during different growth phases, in terms of the growth curve, alterations of taxonomic and functional compositions. The addition of drugs at 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 lag phase may result in a significantly reduced abundance of the Clostridiales order as well as an increased abundance of the Bacteroides genus, which is different from stimulation during the rest of the growth phase. Metformin may also resulte 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.