Assessing the Activity of Single Bacteria through Extracellular Recording of the Dynamical Extrusion of Protons

With the increasing crisis of drug-resistant pathogens, there is an urgent need for the development of rapid, low-invasive, and metabolic activity-oriented single-bacterium viability assessment techniques. In this work, we propose an innovative, proton metabolism tracking-related approach to address this gap in the field that is based on the dynamical tracking of proton extrusion from a single bacterium through confinement-enhanced intracellular pH recording. By confining individual Escherichia coli (E. coli) and less active Bacillus subtilis (B. subtilis) bacteria in a series of microwells preloaded with a pH-sensitive fluorescence probe, we can clearly resolve their different activities in proton metabolism, verifying the feasibility of the proposed method. Furthermore, using E. coli as a model bacterium, we examined the effects of various antibiotics on their proton transport dynamics. The results showed that potassium azide (a respiratory chain inhibitor) and polymyxin (a membrane permeabilizer) rapidly suppressed proton extrusion, whereas ampicillin (a cell wall synthesis inhibitor) exhibited delayed effects due to its indirect action on proton dynamics. These observations are in alignment with the distinct antimicrobial mechanisms, thereby emphasizing the proficiency of the assay in distinguishing the action mechanism of the antibiotics. By transcending population averaging and directly linking extracellular proton dynamics to bacterial activity, this platform facilitates rapid proton metabolism assessment-based bacterial viability analysis, thereby expediting antibiotic susceptibility testing (AST) and drug development. This platform thus offers a promising avenue for screening antibiotics targeting proton transport pathways, a pivotal frontier in the combating of antimicrobial resistance.