From the Microbiome to the Electrome: Implications for the Microbiota-Gut-Brain Axis

Abstract

The gut microbiome plays a fundamental role in metabolism, as well as the immune and nervous systems. Microbial imbalance (dysbiosis) can contribute to subsequent physical and mental pathologies. As such, interest has been growing in the microbiota-gut-brain brain axis and the bioelectrical communication that could exist between bacterial and nervous cells. The aim of this study was to investigate the bioelectrical profile (electrome) of two bacterial species characteristic of the gut microbiome: a Proteobacteria Gram-negative bacillus Escherichia coli (E. coli), and a Firmicutes Gram-positive coccus Enterococcus faecalis (E. faecalis). We analyzed both bacterial strains to (i) validate the fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), as a reliable reporter of the changes in membrane potential (Vmem) for both bacteria; (ii) assess the evolution of the bioelectric profile throughout the growth of both strains; (iii) investigate the effects of two neural-type stimuli on Vmem changes: the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA); (iv) examine the impact of the bioelectrical changes induced by neurotransmitters on bacterial growth, viability, and cultivability using absorbance, live/dead fluorescent probes, and viable counts, respectively. Our findings reveal distinct bioelectrical profiles characteristic of each bacterial species and growth phase. Importantly, neural-type stimuli induce Vmem changes without affecting bacterial growth, viability, or cultivability, suggesting a specific bioelectrical response in bacterial cells to neurotransmitter cues. These results contribute to understanding the bacterial response to external stimuli, with potential implications for modulating bacterial bioelectricity as a novel therapeutic target.

Keywords: Gram-negative; Gram-positive; bis-(1,3-dibutylbarbituric acid) trimethine oxonol-DiBAC; growth phase; membrane potential; microbiota–gut–brain axis; neurotransmitters.

Figure 1

DIBAC validation as a tool for measuring Vmem in E. coli and E. faecalis. (A) Conceptual schematic of DiBAC validation assay. Increasing concentrations of KCl (in presence of valinomycin, Val) were added in the extracellular medium to induce depolarization (due to a lower efflux of K⁺ ions). Created with BioRender.com (Toronto, ON Canada) (B) Quantification of DiBAC fluorescence using an ImageJ macro (National Institutes of Health, Bethesda, MD, USA) . Comparation of percentage of depolarized cells in presence of different KCl concentrations (control or 0 mM, 15 mM, or 60 mM), applying the generalized estimating equations (GEEs) statistical method. A significant increase in the percentage of depolarized cells was observed as the KCl increased in the extracellular medium. *** p-values (p < 0.01). For each experimental condition, values from three biological replicates (dots) with at least three technical replicates each are plotted. (C) Epifluorescence microscopy images. High-magnification images show DiBAC-expressing cells (in green) of E. coli (a–c) and E. faecalis (d–f)) for control (a,d), KCl 15 mM (b,e), and KCl 60 mM (c,f). Scale bar = 10 µM.

Figure 2

Bioelectrical profile throughout bacterial growth. Axenic cultures of E. coli or E. faecalis were prepared in fresh TSB medium and incubated for 5 h. At t = 1 h, t = 3 h, and t = 5 h, bacterial cells were sampled and stained with DiBAC to reveal the membrane potential (Vmem) by epifluorescence microscopy. Generalized estimating equations (GEEs) were applied as the statistical method. (A) Percentage of depolarized bacteria. Values from three biological replicates (dots) with three technical replicates for each condition are represented per time. The percentage of depolarized cells decreased significantly with growth time in E. coli, and, on the contrary, it increased in E. faecalis (*** p < 0.001). The percentage of depolarized cells in E. coli varied from 37.48 ± 2.45% at t = 1 h to 12.18 ± 3.61% at t = 3 h and further reduced to 6.33 ± 1.34% at t = 5 h. Simultaneously, values for growth rate of E. coli population at each time point were rt = 1.34, 0.65, and 0.07 at 1, 3, and 5 h, respectively. The percentage of depolarized cells in E. faecalis varied from 28.31 ± 0.81% at t = 1 h to 43.22 ± 6.62% at t= 3 h and further to 57.27 ± 1.58% at t = 5. Values for growth rate of E. faecalis culture at each time point were rt= 1.05, 0.81, and 0.28 at 1, 3, and 5 h, respectively (for detailed statistics, see Supplementary Table S2). (B) Frequency distribution histograms of DIBAC expression in both bacteria. Data are plotted as the total number of cells, normalized to the number of those exhibiting the most frequent intensity value at each of the three time points. Depolarization threshold (average DiBAC fluorescence intensity at t = 1 h) was set at 65.32 and 99.82 arbitrary units (a.u) for E. coli and E. faecalis, respectively (dashed line).

Figure 3

The effect of neurotransmitters on bacteria bioelectricity. Axenic cultures of E. coli or E. faecalis were prepared in fresh TSB medium and incubated for 4.5 h without (control) or with neurotransmitters (75 µM glutamate or 0.01 µM GABA). Subsequently, the bioelectrical activity of the bacteria in each group was measured using DiBAC as a membrane potential (Vmem) reporter. Analysis by epifluorescence microscopy was made, and generalized estimating equations (GEEs) were applied as the statistical method. (A) Percentage of depolarized bacteria. Values from three biological replicates (dots) with three technical replicates for each condition are represented per time. Both neurotransmitters induced a significant decrease in the percentage of depolarized E. coli or E. faecalis compared to the control (*** p < 0.001). (B) Frequency distribution histograms of DIBAC expression in both bacteria. Data are plotted as the total number of cells, normalized to the number of those exhibiting the most frequent intensity value in each treatment (control, blue; glutamate, orange; GABA, pink). Depolarization threshold (average DiBAC fluorescence intensity value of control cells) was setting at 119.75 and 96 arbitrary units (a.u) for E. coli and E. faecalis, respectively (dashed line).

Figure 4

The effect of neurotransmitters on bacterial growth, cultivability, and viability. (A) Both bacterial strains were incubated with glutamate and GABA for 6 h, during which their growth dynamics were analyzed by measuring optical density at 600 nm (OD600). (B) The cultivability was assessed by viable count (CFU/mL). (C) Bacterial viability was determined using a LIVE/DEAD™ BacLight™ Bacterial Viability Kit to count live and dead cells. Data from three biological replicates (dots) with three technical replicates for each condition are presented for each time point. Neither neurotransmitter caused a significant change in OD600, CFU/mL, or the percentage of live/dead cells (p > 0.05).