Membrane potential dynamics unveil the promise of bioelectrical antimicrobial susceptibility Testing (BeAST) for anti-fungal screening.

Membrane potential is a useful marker for antimicrobial susceptibility testing (AST) due to its fundamental roles in cell function. However, the difficulties associated with measuring the membrane potential in microbes restrict its broad application. In this study, we present bioelectrical AST (BeAST) using the model fungus Saccharomyces cerevisiae. Using fluorescent indicators [DiBAC4(3), ThT, and TMRM], we measured plasma and mitochondrial membrane-potential dynamics upon electric stimulation. We find that a 2.5 second electric stimulation induces hyperpolarization of plasma membrane lasting 20 minutes in vital S. cerevisiae, but depolarization in inhibited cells. The numerical simulation of FitzHugh-Nagumo model successfully recapitulates vitality-dependent dynamics. The model also suggests that the magnitude of plasma-membrane potential dynamics (PMD) correlates with the degree of inhibition. To test this prediction and to examine if BeAST can be used for assessing novel anti-fungal compounds, we treat cells with biogenic silver nanoparticles (bioAgNPs) synthesized using orange fruit flavonoids and Fusarium oxysporum. Comparing BeAST with optical density assay alongside various stressors, we show that PMD correlates with the magnitude of growth inhibitions. These results suggest that BeAST holds promise for screening anti-fungal compounds, offering a valuable approach to tackling antimicrobial resistance. IMPORTANCE: Rapid assessment of the efficacy of antimicrobials is important for optimizing treatments, avoiding misuse and facilitating the screening of new antimicrobials. The need for rapid antimicrobial susceptibility testing (AST) is growing with the rise of antimicrobial resistance. Here, we present bioelectrical AST (BeAST). Combining time-lapse microscopy and mathematical modeling, we show that electrically induced membrane potential dynamics of yeast cells correspond to the strength of growth inhibition. Furthermore, we demonstrate the utility of BeAST for assessing antimicrobial activities of novel compounds using biogenic silver nanoparticles.

Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity.

Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and intercellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cellular proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane-potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely Bacillus subtilis and Escherichia coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5-second electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are preexposed to 400 ± 8 nm wavelength light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore, or alcohol. Therefore, electrically induced membrane-potential dynamics offer a reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.