Metabolomics in microbiology: interrogating microbial physiology and interactions

ABSTRACT Metabolomics provides a direct window into microbial function by capturing the small molecules that reflect cellular activity and microbial interactions. Here, we highlight its diverse applications for microbiologists across both in vitro and in vivo models under dynamic biological and nutritional conditions, including variations in community complexity, medium composition, and exposure to exogenous compounds such as antibiotics. Through representative case studies, we demonstrate how metabolomics provides critical insights into microbial physiology and inter-organismal interactions. We also showcase examples of multiomics integration, in which metabolomics is combined with complementary approaches to enhance data interpretation and strengthen biological conclusions. Finally, we discuss key challenges in metabolomics data analysis and highlight emerging strategies to overcome these limitations.