Measuring bacterial oxygen consumption rate to probe metabolic signature and antimicrobial susceptibility

Quantitative metabolic profiling of bacterial communities provides biophysical insight into cellular respiration and energy transduction under controlled environmental conditions. The oxygen consumption rate (OCR) represents a direct, integrative readout of respiratory activity and has emerged as a sensitive phenotypic parameter for characterizing microbial physiology across different metabolic states. Here, we present an experimental protocol for measuring bacterial respiration using the Seahorse XFe96 Analyzer (Agilent), a microplate-based platform that enables high temporal resolution measurements of oxygen flux. Although originally developed for eukaryotic cell cultures, we adapted this technology to bacterial systems, allowing the simultaneous analysis of multiple strains in a medium-throughput format. The 96-well configuration, combined with sequential injection of up to four compounds, including metabolic substrates, stressors, and alternative electron acceptors, enables real-time perturbation of respiration and monitoring of dynamic OCR responses. To investigate bacterial respiration under physiologically and environmentally relevant oxygen tensions, OCR measurements were additionally performed under hypoxic conditions (5% O₂) by integrating the Seahorse XFe96 Analyzer with an oxygen-controlled glove box. This approach permits precise control of oxygen availability and extends OCR analysis to low-oxygen regimes relevant to chronic infections and aquatic or engineered environments. Overall, our results establish a robust and versatile biophysical platform for probing bacterial respiratory dynamics and for phenotypic screening applications.