High-throughput profiling of bacterial respiration using the Resipher reveals functional responses to nutrients and antibiotics.

Oxygen consumption is a direct functional readout of bacterial respiration and metabolic state, yet existing methods for quantifying oxygen dynamics are limited in throughput and temporal resolution. Here, we establish a high-throughput platform for real-time profiling of bacterial respiration by adapting the Resipher, a non-invasive oxygen quantification system, for use in bacterial cultures. Measurements obtained with the Resipher were comparable to those generated using a Clark-type electrode-based high-resolution respirometer, validating its quantitative accuracy. Across Gram-negative (Escherichia coli, Francisella novicida) and Gram-positive (Enterococcus faecalis, Staphylococcus aureus) species, the Resipher generated reproducible measurements under both growth-permissive and growth-limited conditions, enabling assessment of respiration, independent of proliferation. Functional profiling revealed that oxygen consumption responds dynamically to nutrient availability and electron transport chain perturbation, including species-specific inhibition by benzarone. Notably, oxygen consumption profiles distinguished bactericidal and bacteriostatic antibiotics, with bactericidal agents transiently increasing respiration and bacteriostatic agents suppressing metabolic activity. Together, these findings establish oxygen consumption as a sensitive physiological readout and highlight the potential utility of respiratory profiling for mechanistic studies.