Inter-cellular communication is mediated by a sum of biochemical, biophysical, and bioelectrical signals. This might occur not only between cells belonging to the same tissue and/or animal species but also between cells that are, from an evolutionary point of view, far away. The possibility that bioelectrical communication takes place between bacteria and nerve cells has opened exciting perspectives in the study of the gut microbiota–brain axis. The aim of this paper is (i) to establish a reliable method for the assessment of the bioelectrical state of two bacterial strains: Bacillus subtilis (B. subtilis) and Limosilactobacillus reuteri (L. reuteri); (ii) to monitor the bacterial bioelectrical profile throughout its growth dynamics; and (iii) to evaluate the effects of two neurotransmitters (glutamate and γ-aminobutyric acid-GABA) on the bioelectrical signature of bacteria. Our results show that membrane potential (Vmem) and the proliferative capacity of the population are functionally linked in B. subtilis in each phase of the cell cycle. Remarkably, we demonstrate that bacteria respond to neural signals by changing Vmem properties. Finally, we show that Vmem changes in response to neural stimuli are present also in a microbiota-related strain L. reuteri. Our proof-of-principle data reveal a new methodological approach for the better understanding of the relation between bacteria and the brain, with a special focus on gut microbiota. Likewise, this approach will open exciting perspectives in the study of the inter-cellular mechanisms which regulate the bi-directional communication between bacteria and neurons and, ultimately, for designing gut microbiota–brain axis-targeted treatments for neuropsychiatric diseases.

The DNA topoisomerase complement of Streptococcus pneumoniae is constituted by two type II enzymes (topoisomerase IV and gyrase), and a single type I enzyme (topoisomerase I). These enzymes maintain the DNA topology, which is essential for replication and transcription. While fluoroquinolones target the type II enzymes, seconeolitsine, a new antimicrobial agent, targets topoisomerase I. We compared for the first time the in vitro effect of inhibition of topoisomerase I by seconeolitsine and of the type II topoisomerases by the fluoroquinolones levofloxacin and moxifloxacin. We used three isogenic non-encapsulated strains and five non-vaccine serotypes isolates belonging to two circulating pneumococcal clones, ST63⁸ (2 strains) and ST156^9V (3 strains). Each group contained strains with diverse susceptibility to fluoroquinolones. Minimal inhibitory concentrations, killing curves and postantibiotic effects were determined. Seconeolitsine demonstrated the fastest and highest bactericidal activity against planktonic bacteria and biofilms. When fluoroquinolone-susceptible planktonic bacteria were considered, seconeolitsine induced postantibiotic effects (1.00−1.87 h) similar than levofloxacin (1.00−2.22 h), but longer than moxifloxacin (0.39−1.71 h). The same effect was observed in sessile bacteria forming biofilms. Seconeolitsine induced postantibiotic effects (0.84−2.31 h) that were similar to those of levofloxacin (0.99−3.32 h) but longer than those of moxifloxacin (0.89−1.91 h). The greatest effect was observed in the viability and adherence of bacteria in the postantibiotic phase. Seconeolitsine greatly reduced the thickness of the biofilms formed in comparison with fluoroquinolones: 2.91 ± 0.43 μm (seconeolitsine), 7.18 ± 0.58 μm (levofloxacin), 17.08 ± 1.02 μm (moxifloxacin). When fluoroquinolone-resistant bacteria were considered, postantibiotic effects induced by levofloxacin and moxifloxacin, but not by seconeolitsine, were shorter, decreasing up to 5-fold (levofloxacin) or 2-fold (moxifloxacin) in planktonic cells, and up to 1.7 (levofloxacin) or 1.4-fold (moxifloxacin) during biofilm formation. Therefore, topoisomerase I inhibitors could be an alternative for the treatment of pneumococcal diseases, including those caused by fluoroquinolone-resistant isolates.

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.

Scope: Iron deficiency (ID) is the most common nutritional deficiency worldwide, impacting gut bacteria's metabolism and cellular biochemistry, but its effects on the microbiota‐gut‐brain axis (MGB) are poorly understood. Early‐life ID‐related dysbiosis is linked to neurodevelopmental impairments like autism and attention deficit hyperactivity disorder. Studying ID's impact on bacterial signaling can guide interventions to target MGB in iron‐deficient populations. This study examined the responses of Escherichia coli (E. coli) and Limosilactobacillus reuteri (L. reuteri) to in‐vitro ID conditions using the iron chelator 2,2’‐Bipyridyl (BP). Methods and Results: We assessed and modeled their growth and cultivability and explored their bioelectric profiles using the voltage‐sensitive dye DiBAC4(3). Results showed differential responses: L. reuteri's growth and cultivability were unaffected by BP, while E. coli's growth rate and cultivability decreased under ID. Additionally, we created a deterministic mathematical model that demonstrated a decrease in the population's average reproduction rate in E. coli under ID. Only E. coli exhibited an altered bioelectric profile, marked by increased cell depolarization in ID conditions, which was largely rescued upon the addition of a saturating concentration of iron. Conclusion: These findings highlight specific bioelectrical responses in gut bacteria to ID. Understanding this variability is crucial for deciphering the microbiota's role in health and disease, particularly concerning nutritional iron imbalance and bacterial signaling in the MGB.