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Divya Naradasu, Waheed Miran, [Akihiro Okamoto](https://orcid.org/0000-0002-8102-4316)

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[Electrochemical Characterization of Two Gut Microbial Strains Cooperatively Promoting Multiple Sclerosis Pathogenesis](https://mdr.nims.go.jp/datasets/c8bbf4f0-d8ae-429f-b22f-600ce51492a2)

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Electrochemical Characterization of Two Gut Microbial Strains Cooperatively Promoting Multiple Sclerosis PathogenesisCitation: Naradasu, D.; Miran, W.;Okamoto, A. ElectrochemicalCharacterization of Two GutMicrobial Strains CooperativelyPromoting Multiple SclerosisPathogenesis. Microorganisms 2024, 12,257. https://doi.org/10.3390/microorganisms12020257Academic Editor: Nico JehmlichReceived: 21 December 2023Revised: 13 January 2024Accepted: 16 January 2024Published: 25 January 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).microorganismsArticleElectrochemical Characterization of Two Gut Microbial StrainsCooperatively Promoting Multiple Sclerosis PathogenesisDivya Naradasu 1,2,†, Waheed Miran 2,3,† and Akihiro Okamoto 2,4,5,6,7,*1 Oral Microbiology, Bristol Dental School, University of Bristol, Dorothy Hodgkin Building,Whitson Street, Bristol BS1 3NY, UK; divya.naradasu@bristol.ac.uk2 International Center for Materials Nanoarchitectonics, National Institute for Materials Science,1-1 Namiki, Tsukuba 305-0044, Ibaraki, Japan; waheed.miran@scme.nust.edu.pk3 School of Chemical and Materials Engineering, National University of Sciences and Technology,Islamabad 44000, Pakistan4 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science,Tsukuba 305-0044, Ibaraki, Japan5 Graduate School of Chemical Sciences and Engineering, Hokkaido University, North 13 West 8,Kita-ku, Sapporo 060-8628, Hokkaido, Japan6 Graduate School of Science and Engineering, College of Science and Engineering, University of Tsukuba,1-1-1 Tennodai, Tsukuba 305-8573, Ibaraki, Japan7 Living Systems Materialogy (LiSM) Research Group, International Research Frontiers Initiative (IRFI),Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Kanagawa, Japan* Correspondence: okamoto.akihiro@nims.go.jp† These authors contributed equally to this work.Abstract: In this study, we explored the extracellular electron transfer (EET) capabilities of two bac-terial strains, OTU0001 and OTU0002, which are demonstrated in biofilm formation in mouse gutand the induction of autoimmune diseases like multiple sclerosis. OTU0002 displayed significantelectrogenic behaviour, producing microbial current on an indium tin-doped oxide electrode sur-face, particularly in the presence of glucose, with a current density of 60 nA/cm2. The presence ofcell-surface redox substrate potentially mediating EET was revealed by the redox-based stainingmethod and electrochemical voltammetry assay. However, medium swapping analyses and theaddition of flavins, a model redox mediator, suggest that the current production is dominated bysoluble endogenous redox substrates in OTU0002. Given redox substrates were detected at the cellsurface, the secreted redox molecule may interact with the cellular surface of OTU0002. In contrastto OTU0002, OTU0001 did not exhibit notable electrochemical activity, lacking cell-surface redoxmolecules. Further, the mixture of the two strains did not increase the current production fromOTU0001, suggesting that OTU0001 does not support the EET mechanism of OTU0002. The presentwork revealed the coexistence of EET and non-EET capable pathogens in multi-species biofilm.Keywords: whole-cell electrochemistry; gut microbes; extracellular electron transport; multiplesclerosis1. IntroductionThe field of microbiology has seen significant advancements in understanding theextracellular electron transfer (EET) mechanism, particularly in environmental bacteria.These bacteria transfer electrons to extracellular solids like Fe (III) and Mn (IV) duringanaerobic respiration [1–3]. This process, crucial for their metabolic activities, involveseither direct or indirect electron transfer mechanisms [4,5]. In the direct EET mechanisms,c-type cytochrome complexes, which are multiheme proteins, are integral in transport-ing electrons across the outer membrane of these bacteria. This has been particularlywell-documented in strains like Shewanella oneidensis MR-1 and Geobacter sulfurreducensPCA. These outer membrane cytochromes (OMCs) play a pivotal role in a wide arrayMicroorganisms 2024, 12, 257. https://doi.org/10.3390/microorganisms12020257 https://www.mdpi.com/journal/microorganismshttps://doi.org/10.3390/microorganisms12020257https://doi.org/10.3390/microorganisms12020257https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/microorganismshttps://www.mdpi.comhttps://orcid.org/0000-0002-8380-1537https://orcid.org/0000-0002-8102-4316https://doi.org/10.3390/microorganisms12020257https://www.mdpi.com/journal/microorganismshttps://www.mdpi.com/article/10.3390/microorganisms12020257?type=check_update&version=1Microorganisms 2024, 12, 257 2 of 11of electroactive microbes [4,6–8]. Notably, a few micromolars of endogenous riboflavin(RF) and flavin mononucleotide (FMN) significantly boost the rate of direct EET via theOMCs as a non-covalently bound cofactor [9,10]. The indirect EET mechanism can beenhanced by both endogenous and exogenous redox mediators. These mediators facili-tate electron shuttling between microbes and electrodes, governed by diffusion-limitedkinetics [11,12]. Therefore, indirect EET mechanisms usually require concentrations ofaround 100 micromolars of the soluble redox mediator to achieve a similar level of currentenhancement with the bound flavins [9,13]. The occurrence of these EET mechanisms hasbeen recently uncovered in diverse and unique ecological niches, including those involvinghuman-to-animal pathogens [14,15].Certain well-known human pathogens exhibit electrogenic behaviour, suggesting a linkbetween their physiology and electrochemical activities. For instance, Listeria monocytogenes,a human gut-associated pathogen, can produce an electric current, which correlates withthe extent of biofilm formation [14,16]. Further, Enterococcus faecalis [17,18] and a couple ofhuman gut-isolated strains that have high genetic similarity with Klebsiella pneumoniae andEnterococcus avium are capable of EET [19]. Pseudomonas aeruginosa, one of the key playersin human infections, is known to secrete high concentrations of phenazines for indirect EETmechanism [20]. Each EET mechanism is suggested to be involved in microbial physiology,facilitating the bacterium’s growth and pathogenicity within the host environment.Consistent with such ideas, pathogens from oral biofilm have the EET capabilityand potentially make a thermodynamically downhill electron transport pathway withina 100 µm polymicrobial biofilm structure [15,21]. These microorganisms, within polymi-crobial aggregates, are known to engage in interspecies electron transfer (IET), in addi-tion to hydrogen or formate transfer, to boost their metabolism under anaerobic condi-tions [22,23]. Streptococcus mutans, Corynebacterium matruchotii, Porphyromonas gingivalis,and Aggregatibacter actinomycetemcomitans produce electric current associated with thepresence of the cell-surface redox agents. The energy diagrams for these redox agentsindicate a redox potential cascade from the anaerobic core to the periphery of the biofilm.This suggests that an IET may play a key role in supporting microbial metabolism in theanaerobic regions of the biofilm [15]. Understanding the mechanisms of IET is thereforevital for managing the activities of complex polymicrobial biofilms. However, the pres-ence of multiple species complicates this task, requiring experimental approaches beyondomics studies, such as metatranscriptomics, due to their intricate nature [24]. A recentstudy identified the biofilm formation of two gut microbial strains, phylogenetically simi-lar to Lactobacillus reuteri (OTU0001) and Allobaculum stercoricanis DSM 13633 (OTU0002),cooperatively induce multiple sclerosis (MS), an autoimmune disorder [25]. While theEET-capability of these strains has not been studied, the two-strain-biofilm system could bea model system for studying the interspecies electron transfer in biofilm formation and itspotential impact on host-microbe interaction.The present study investigated the EET capability of two gut microbes (OTU0002 andOTU0001). Using techniques such as single potential amperometry and differential pulsevoltammetry, we aim to reveal the specific EET activities of these microbes. Our exper-imental approach, as shown in the schematic of the overall study mechanism in FigureS1, involves investigating direct and mediated current EET through supernatant replace-ment in a three-electrode chemical reactor, providing a thorough analysis of the electricalcharacteristics associated with these microbial strains.2. Materials and Methods2.1. Cell Culture PreparationPure strains of operational taxonomic unit (OTU) 0001 and 0002, obtained from RIKEN,Japan, were pre-cultured in a sterilized LB medium aerobically. All cultures were incubatedat 37 ◦C until they reached the late exponential phase of growth. The cells were thencentrifuged at 7200 rpm and 37 ◦C for 10 min. The resulting cell pellet was washed twicewith a defined medium (DM) to eliminate any cell-secreted metabolites from the previousMicroorganisms 2024, 12, 257 3 of 11culture medium. The cell cultures were handled in a COY anaerobic chamber filled with100% nitrogen. The DM was prepared using previously described methods [26] withsome modifications. DM contained (per Liter) the following components: NaHCO3, 2.5 g;CaCl2·2H2O, 0.09 g; NH4Cl, 1.0 g; MgCl2·6H2O, 0.2 g; NaCl, 10 g; HEPES, 7.2 g.2.2. Transmission Electron MicroscopyTo collect cells at the exponential growth phase, 2–4 mL of 20 mL pre-cultures werecentrifuged at 6000 rpm for 10 min. The cells were then immediately fixed in solutionscontaining 2% paraformaldehyde and 2.5% glutaraldehyde on ice. All further manipula-tions were conducted in 2 mL Eppendorf tubes after fixation. The washing process wasdone in 50 mM Na+-HEPES (pH 7.4, 35 g/L NaCl) with 5 × 1.5-mL washes by gently resus-pending the pellet and centrifugation (5000× g, 4 min). Sequential metal enzyme-reactive3, 3′-diaminobenzidine −H2O2 staining, OsO4 staining, and resin embedding procedureswere conducted by following the previously described method [23]. The obtained resinblocks were sectioned at 80 nm with a diamond knife (DiATOME, ultra 35◦), and floatingsections were mounted on copper microgrids (Nishan EM). Thin sections were examinedand imaged using a JEM-1400 microscope operated at 80 kV.2.3. Whole-Cell Electrochemical Analysis of Pure StrainsElectrochemical measurements were conducted in single-chamber, three-electrodereactors. Indium tin-doped oxide (ITO) grown on a glass substrate by spray pyrolysisdeposition (SPD Laboratory, Inc., Hamamatsu, Japan) was used as the working electrode(resistance, 8 Ω/square; thickness, 1.1 mm; and surface area, 3.14 cm2) and was placed atthe bottom of the reactor. Platinum wire and Ag/AgCl (sat. KCl) were used as counterand reference electrodes, respectively. DM containing 10 mM glucose (4.8 mL) was injectedinto the electrochemical cell as an electrolyte, and the solution was purged with N2 gasfor at least 15 min to remove the dissolved oxygen. The electrochemical cell was thenfilled with 0.2 mL of fresh cell suspension of OTU0001 and OTU0002, prewashed withDM, and injected to a final optical density of 0.5 and 1 at 600 nm under a potentiostaticcondition of +0.4 V vs. the standard hydrogen electrode (SHE). During the electrochemicalmeasurements, the reactor temperature was maintained at 37 ◦C, the reactor was operatedwithout agitation, and experiments were run for about 24 h. Single-potential amperometryand differential pulse voltammetry (DPV) were measured with an automatic polarizationsystem (VMP3, BioLogic Company, Seyssinet-Pariset, France). DPV was measured underthe following conditions: from −0.6 V to +0.7 V vs. SHE at a pulse height of 50 mV, pulsewidth of 0.3 s, step height of 5 mV s−1, and step time of 5 s [9].2.4. Supernatant Exchange Experiments during the Current Producing ConditionThe medium in the electrochemical cell was removed; the electrode-attached cellswere rinsed with N2-sparged DM twice at each replacement; and the headspace wascontinuously sparged with N2 during each replacement to avoid the leakage of oxygeninto the electrochemical cell. The cell was refilled with N2-sparged sterile medium (10 mMglucose) and supernatant containing either planktonic cells or filtered medium.2.5. Scanning Electron Microscopy (SEM)For the scanning electron microscopy, ITO electrodes were removed from the reactorsafter performing the electrochemical measurements for about 24 h and slightly washed with0.1 M phosphate buffer to remove the cells suspended on the electrode surface. Microbialfixation on electrodes was carried out with 2.5% glutaraldehyde diluted in 0.1 M phosphatebuffer for more than one hour in the dark at room temperature. This was followed bywashing three times in 0.1 M phosphate buffer (pH 7.4) for 15 min each. These washedsamples were then dehydrated in 25%, 50%, 75%, and 100% ethanol gradients for 15 mineach. Ethanol gradient dehydrated samples were exchanged thrice with 100% t-butanoland finally freeze-dried under a vacuum. The dried samples were coated with platinumand then observed using a Keyence VE-9800 microscope.Microorganisms 2024, 12, 257 4 of 113. Results and Discussion3.1. Electrochemical Characterization of OTU0001 and OTU0002To evaluate the electron transfer abilities of gut pathogens OTU0002 and OTU0001,we examined their EET capability by measuring current production (ia) via single-potentialamperometry with the ITO working electrode poised at +0.4 V vs. SHE. Following theintroduction of bacterial cells into the reactor, a significant rise in net current of about60 nA cm−2 was noted at a final optical density of 0.5 at 600 nm (OD600 nm). This increaseimmediately occurred in the presence of 10 mM glucose (blue line, Figure 1A). In contrast,no significant current was produced in the presence of OTU0001 (red line, Figure 1A) andthe absence of microbes (black line, Figure 1A). These findings clearly imply that OTU0002’sEET capacity in conjunction with glucose oxidation.Microorganisms 2024, 12, x FOR PEER REVIEW  4  of  13   washed samples were then dehydrated in 25%, 50%, 75%, and 100% ethanol gradients for 15 min each. Ethanol gradient dehydrated samples were exchanged thrice with 100% t-butanol and finally freeze-dried under a vacuum. The dried samples were coated with platinum and then observed using a Keyence VE-9800 microscope. 3. Results and Discussion 3.1. Electrochemical Characterization of OTU0001 and OTU0002 To evaluate the electron transfer abilities of gut pathogens OTU0002 and OTU0001, we examined their EET capability by measuring current production (ia) via single-poten-tial amperometry with the ITO working electrode poised at +0.4 V vs. SHE. Following the introduction of bacterial cells into the reactor, a significant rise in net current of about 60 nA cm−2 was noted at a final optical density of 0.5 at 600 nm (OD600 nm). This increase immediately occurred in the presence of 10 mM glucose (blue line, Figure 1A). In contrast, no significant current was produced in the presence of OTU0001 (red line, Figure 1A) and the  absence  of  microbes  (black  line,  Figure  1A).  These  findings  clearly  imply  that OTU0002’s EET capacity in conjunction with glucose oxidation.  Figure 1. Evidence for extracellular electron transfer by the gut pathogen OTU0002. (A) Time vs. Current  profile  during  the  Single-potential  amperometry  (SA)  over  time  in  anaerobic  reactors equipped with ITO electrodes at +0.4 V (SHE) in the presence of 10 mM glucose. OTU0002 showed a significant current production (blue line) compared to OTU0001 (red line) and without any mi-crobes in the reactor (black line). (B). Differential pulse voltammograms in the presence of OTU0002 (blue line) and OTU0001 (red line). Data for sterile DM without any added microbes (black line) are also rep-resented. (C,D) After 24 h of producing current with 10 mM glucose at +0.4 V (versus SHE), intact cells were observed attached to the electrode surface in scanning electron microscope images of OTU0002. After stabilizing the current production, a differential pulse voltammogram was con-ducted to investigate the electrochemical properties of the cell suspension. The presence of OTU0002 resulted in a broad oxidative peak ranging from approximately −0.3 V to ~+0.1 V vs. SHE, with the maximum peak potential (Ep) at around −0.13 V (SHE) (as shown in Figure 1B).The large half-peak width of more than 200 mV (vs. SHE) is consistent with Figure 1. Evidence for extracellular electron transfer by the gut pathogen OTU0002. (A) Time vs.Current profile during the Single-potential amperometry (SA) over time in anaerobic reactors equippedwith ITO electrodes at +0.4 V (SHE) in the presence of 10 mM glucose. OTU0002 showed a significantcurrent production (blue line) compared to OTU0001 (red line) and without any microbes in the reactor(black line). (B). Differential pulse voltammograms in the presence of OTU0002 (blue line) and OTU0001(red line). Data for sterile DM without any added microbes (black line) are also represented. (C,D) After24 h of producing current with 10 mM glucose at +0.4 V (versus SHE), intact cells were observed attachedto the electrode surface in scanning electron microscope images of OTU0002.After stabilizing the current production, a differential pulse voltammogram wasconducted to investigate the electrochemical properties of the cell suspension. The presenceof OTU0002 resulted in a broad oxidative peak ranging from approximately −0.3 V to~+0.1 V vs. SHE, with the maximum peak potential (Ep) at around −0.13 V (SHE) (asshown in Figure 1B).The large half-peak width of more than 200 mV (vs. SHE) is consistentwith multi-heme outer-membrane cytochromes but not with soluble electron mediatorssuch as riboflavin with that of 60 mV (vs. SHE) [27]. DP voltammetry did not showany significant peak current in the absence of microbes or the presence of OTU0001 (asshown in Figure 1B, black and red line). After several washes and dehydration, cellularattachment was observed on the ITO electrode surface for both strains using scanningMicroorganisms 2024, 12, 257 5 of 11electron microscopy (Figure 1C,D). This implies that the voltammetric signal is a result ofthe presence of redox enzymes on the surface of the microbe of OTU0002.The assignment of the broad redox signal was supported by the change of currentproduction upon electrolyte exchange experiments (Figure S2A). Even after supernatanttransfer to a fresh medium, a similar peak current with a broad potential window wasobserved on DP voltammograms (Figure S2B, black and red lines), indicating that exoge-nous or endogenous soluble redox-active compounds are not the primary components ofthe redox signal. Therefore, the redox substrates on the cell surface are the most probablecandidates attributable to the electrochemical signal. While DPV of spent cell-free spentmedium exchange resulted in a slightly shifted peak potential (−0.14 V) and a second peakat −0.26 V, distinct from the main peak at −0.14 V solely coming from the cells remained onthe electrode surface after fresh medium exchange (Figure S2B, red line). The electrochemi-cal redox peak might be attributed to proteins that have numerous redox molecules withvarying redox potentials, as the combinatorial overlap of multi-redox processes increasesthe apparent peak breadth, resulting in a broad peak appearance.We also performed an in vitro analysis utilizing the redox-dependent DAB chemicalstaining technique to identify cell-surface redox chemicals [23]. The two bacterial strains,OTU0002 and OTU0001, were grown under the same conditions and stained with DAB, andcross-sections were imaged under a transmission electron microscope (TEM). When DABwas negative, the cross-sectional images showed that OTU0002 cells stained with DAB didnot exhibit any black precipitate (Figure 2A). Nonetheless, there was a noticeable distinctionwith thicker cellular margins in the presence of H2O2 (DAB positive, Figure 2B) as opposedto the absence of H2O2. On the other hand, the DAB-negative and -positive images ofOTU0001 exhibited minimal black precipitation on the stained membrane (Figure 2C,D).These data suggest the presence and absence of redox substrate on the cell surface inOTU0002 and 0001, respectively, consistent with electrochemical analysis.Microorganisms 2024, 12, x FOR PEER REVIEW  6  of  13    Figure 2. OTU0002 and OTU0001 cells stained with 3,3′-diaminobenzidene (DAB) are shown in a transmission electron microscopy (TEM) (A) and Positive DAB staining with the addition of H2O2 (B). Negative DAB stained OTU0001 in the absence and presence of H2O2 (C,D), respectively. Scale bars are shown in the images. CW: cell wall; PS: Periplasmic space; PM: Plasma membrane. It  is  expected  that  cell-surface  redox  proteins  would  be  expressed  given  that OTU0002 showed evidence of current production capability (Figure 1A). Thus, the redox staining seen on the cellular membrane further proved that OTU0001 was not capable of transferring electrons or that it has a redox centre that is less reactive to H2O2 oxidation. Our findings suggest that OTU0002 has extracellular or outer membrane proteins with metal centres that can catalyse H2O2 oxidation since the DAB process  involves reaction centres that contain transition metals [28]. Therefore, a comprehensive analysis of cell sur-face proteins is necessary to identify the membrane proteins in these disorders, and this will be a critical area of focus for the future of our studies. Although the electrochemical characterization was conducted without identifying EET genes, our data on DAB suggest that it may be worthwhile to utilize gene expression analysis and gene-deletion techniques in future studies. This is because no EET genes have been identified in OTU0002 in the current literature [14]. To examine the coupling of metabolism with microbial current production, we ana-lysed the activity of cells directly attached to the electrode during EET with Nano-SIMS by measuring the uptake of 15N (the only nitrogen source) by individual cells that most likely contributed  to  the current production  [23,29]. After electrochemical experiments, we rigorously washed the electrode surface to remove planktonic or weakly attached cells on the electrode to analyse the anabolic 15N assimilation of cells by measuring 15N/Ntotal (%) and subtracting  the natural abundance of  15N.  In  the single-potential amperometry Figure 2. OTU0002 and OTU0001 cells stained with 3,3′-diaminobenzidene (DAB) are shown in atransmission electron microscopy (TEM) (A) and Positive DAB staining with the addition of H2O2 (B).Negative DAB stained OTU0001 in the absence and presence of H2O2 (C,D), respectively. Scale barsare shown in the images. CW: cell wall; PS: Periplasmic space; PM: Plasma membrane.It is expected that cell-surface redox proteins would be expressed given that OTU0002showed evidence of current production capability (Figure 1A). Thus, the redox stainingMicroorganisms 2024, 12, 257 6 of 11seen on the cellular membrane further proved that OTU0001 was not capable of transferringelectrons or that it has a redox centre that is less reactive to H2O2 oxidation. Our findingssuggest that OTU0002 has extracellular or outer membrane proteins with metal centresthat can catalyse H2O2 oxidation since the DAB process involves reaction centres thatcontain transition metals [28]. Therefore, a comprehensive analysis of cell surface proteinsis necessary to identify the membrane proteins in these disorders, and this will be a criticalarea of focus for the future of our studies. Although the electrochemical characterizationwas conducted without identifying EET genes, our data on DAB suggest that it may beworthwhile to utilize gene expression analysis and gene-deletion techniques in futurestudies. This is because no EET genes have been identified in OTU0002 in the currentliterature [14].To examine the coupling of metabolism with microbial current production, we anal-ysed the activity of cells directly attached to the electrode during EET with Nano-SIMSby measuring the uptake of 15N (the only nitrogen source) by individual cells that mostlikely contributed to the current production [23,29]. After electrochemical experiments, werigorously washed the electrode surface to remove planktonic or weakly attached cells onthe electrode to analyse the anabolic 15N assimilation of cells by measuring 15N/Ntotal (%)and subtracting the natural abundance of 15N. In the single-potential amperometry con-dition, OTU0002 under the current production condition presented a significantly higher15N (0.4%) anabolic activity during EET (Figure 3A). In contrast, the 15N assimilation wasalmost tenfold smaller in the OCV condition (Figure 3B,C). This distinct anabolic metabolicactivity demonstrates the coupling with the current production of OTU0002.Microorganisms 2024, 12, x FOR PEER REVIEW  7  of  13   condition, OTU0002 under  the  current  production  condition  presented  a  significantly higher 15N (0.4%) anabolic activity during EET (Figure 3A). In contrast, the 15N assimilation was almost tenfold smaller in the OCV condition (Figure 3B,C). This distinct anabolic met-abolic activity demonstrates the coupling with the current production of OTU0002.  Figure 3. NanoSIMS  images of OTU0002 cells attached  to electrodes after EET  (A) and OCV  (B) measurements show the 12C14N− and 12C15N− ion pixel intensities. scale bar = 5 µm. The colour gradi-ent bar indicates ion pixel intensity. Arrows indicate the cells. (C) Average assimilation with ± stand-ard error mean (SEM) of 15N% assimilation of OTU0002 cells under EET and OCV, Number of cells selected for assimilation analysis, n = 64 and 61 for EET and OCV, respectively. 3.2. Potential Involvement of Exogenous and Endogenous Redox Mediators on the Microbial Current Production of OTU0002 To investigate the EET capability of OTU0002, we examined the effect of cell density on its current production. We found that at ODs of 0.1 and 0.2, there was no significant increase  in  current production  (Figure 4A). However, when  the  cell density was at an OD600 of 0.5, the current production increased by approximately 4 times compared to the OD600 of 0.1. Moreover, even though the cell density was enough to cover the whole elec-trode surface at an OD600 of 0.5 (Figure 4A), the current production was higher (~173 nA cm−2) at an OD600 of 1.0, indicating a non-linear relationship with cell density (Figure S3). It is important to note that when the electrode surface is fully covered at OD600 0.5, the microbial current production is not only from cells attached to the electrode surface but also from those without direct attachment. To quantify the contribution of planktonic cells in current production, we swapped the medium with the cell-removed filtered supernatant collected from a reactor (Figure S2A). The current production did not increase and stayed at a low level. Given planktonic cells were also removed from the reactor and some cells should be attached to the elec-trode as SEM represents, this result indicates the dominant contribution of planktonic cells on the current production by OTU0002. The OTU0002 transfers electrons either through accumulative redox mediators in the supernatant or intercellular electron transfer via di-rect electron exchange among the cell-surface redox reagents [30]. Figure 3. NanoSIMS images of OTU0002 cells attached to electrodes after EET (A) and OCV (B)measurements show the 12C14N− and 12C15N− ion pixel intensities. scale bar = 5 µm. The colourgradient bar indicates ion pixel intensity. Arrows indicate the cells. (C) Average assimilation with± standard error mean (SEM) of 15N% assimilation of OTU0002 cells under EET and OCV, Numberof cells selected for assimilation analysis, n = 64 and 61 for EET and OCV, respectively.3.2. Potential Involvement of Exogenous and Endogenous Redox Mediators on the MicrobialCurrent Production of OTU0002To investigate the EET capability of OTU0002, we examined the effect of cell densityon its current production. We found that at ODs of 0.1 and 0.2, there was no significantincrease in current production (Figure 4A). However, when the cell density was at an OD600Microorganisms 2024, 12, 257 7 of 11of 0.5, the current production increased by approximately 4 times compared to the OD600of 0.1. Moreover, even though the cell density was enough to cover the whole electrodesurface at an OD600 of 0.5 (Figure 4A), the current production was higher (~173 nA cm−2)at an OD600 of 1.0, indicating a non-linear relationship with cell density (Figure S3). It isimportant to note that when the electrode surface is fully covered at OD600 0.5, the microbialcurrent production is not only from cells attached to the electrode surface but also fromthose without direct attachment.Microorganisms 2024, 12, x FOR PEER REVIEW  8  of  13    Figure 4. Population-induced EET capability and mediating electron transfer of OTU0002. (A) Cur-rent measurements in anaerobic reactors with ITO electrodes (3.14 cm2) at +0.2 V vs. Ag/AgCl in the presence of 10 mM glucose and OD600. 0.1, 0.2, 0.5, and 1.0 represent the initial cell densities added to the reactors. The arrow position indicates the time of cell addition in the electrochemical reactor. (B) Current production by OTU0002  in  the absence  (control) and  the presence of external  redox active additives, i.e., Riboflavin (RF) and Flavin mononucleotide (FMN). The arrow position indi-cates the time of mediator addition in the electrochemical reactor. (C,D) Differential pulse (DP) volt-ammogram of OTU0002 in the presence and absence (control) of redox-active additives (RF, FMN). We investigated the impact of external redox-active additives, such as Riboflavin (RF) and Flavin mononucleotide (FMN), which are ubiquitous exogenous redox substrates in gut environments [31,32], on current production (Figure 4B). The addition of RF and FMN to the reactor increased current production over threefold compared to the absence of re-dox-active additives (Figure 4B). In addition to Ep of 0.10 V vs. SHE assignable to the cell surface protein, sharp oxidative peaks assignable to free RF were observed with Ep at 0.23 V (SHE) in the presence of RF in the DP voltammogram (Figure 4C). In the case of FMN, the cell-specific redox peak showed a shift towards positive potential at +0.1 V vs. SHE besides a sharp peak rise at ~−0.25 V vs. SHE specific to FMN. Therefore, these data sug-gest OTU0002 uses free FMN substrate as an electron shuttle (Figure 4D). Given that OTU0001 does not have EET capability, we hypothesized that OTU0001 may produce  redox  active  substrates  to  accelerate  the metabolic  activity  of OTU0002. However, OTU0001 and 0002 did not show enhanced current production upon mixing two species in a single EC reactor (Figure S4). Contrary to the synergetic effect on multiple sclerosis by combinational OTU0001 and 2 in our electrochemical analysis, the mixture of equal cell densities of OTU0002 and OTU0001 in the EC reactor showed a decreased cur-rent production (~35 nA/cm2, Figure S4) compared to OTU0002 alone current production, implicating a non-synergetic dependency with each other and absence of Quorum sensing (QS) mediated  communication,  thus  electron  transfer  reduced  to half. However,  these data clearly indicate the impact of OTU0001 on OTU0002 either by diminishing its activity or sharing the metabolic electrons. The decrease could be due to OTU0001’s antimicrobial activity as L. reuteri, which is similar, produces antimicrobial molecules that inhibit harm-ful microbes, and change the host’s microbiota composition [33]. Given, electron mediators affect  interactions with the bacterial surface, eventually, the transfer of electrons can be enhanced to maintain redox homeostasis within cells. [34]. Figure 4. Population-induced EET capability and mediating electron transfer of OTU0002. (A) Currentmeasurements in anaerobic reactors with ITO electrodes (3.14 cm2) at +0.2 V vs. Ag/AgCl in thepresence of 10 mM glucose and OD600. 0.1, 0.2, 0.5, and 1.0 represent the initial cell densities added to thereactors. The arrow position indicates the time of cell addition in the electrochemical reactor. (B) Currentproduction by OTU0002 in the absence (control) and the presence of external redox active additives, i.e.,Riboflavin (RF) and Flavin mononucleotide (FMN). The arrow position indicates the time of mediatoraddition in the electrochemical reactor. (C,D) Differential pulse (DP) voltammogram of OTU0002 in thepresence and absence (control) of redox-active additives (RF, FMN).To quantify the contribution of planktonic cells in current production, we swapped themedium with the cell-removed filtered supernatant collected from a reactor (Figure S2A).The current production did not increase and stayed at a low level. Given planktonic cellswere also removed from the reactor and some cells should be attached to the electrodeas SEM represents, this result indicates the dominant contribution of planktonic cells onthe current production by OTU0002. The OTU0002 transfers electrons either throughaccumulative redox mediators in the supernatant or intercellular electron transfer via directelectron exchange among the cell-surface redox reagents [30].We investigated the impact of external redox-active additives, such as Riboflavin (RF)and Flavin mononucleotide (FMN), which are ubiquitous exogenous redox substrates ingut environments [31,32], on current production (Figure 4B). The addition of RF and FMNto the reactor increased current production over threefold compared to the absence ofredox-active additives (Figure 4B). In addition to Ep of −0.10 V vs. SHE assignable to thecell surface protein, sharp oxidative peaks assignable to free RF were observed with Ep at−0.23 V (SHE) in the presence of RF in the DP voltammogram (Figure 4C). In the case ofFMN, the cell-specific redox peak showed a shift towards positive potential at +0.1 V vs.Microorganisms 2024, 12, 257 8 of 11SHE besides a sharp peak rise at ~−0.25 V vs. SHE specific to FMN. Therefore, these datasuggest OTU0002 uses free FMN substrate as an electron shuttle (Figure 4D).Given that OTU0001 does not have EET capability, we hypothesized that OTU0001may produce redox active substrates to accelerate the metabolic activity of OTU0002.However, OTU0001 and 0002 did not show enhanced current production upon mixingtwo species in a single EC reactor (Figure S4). Contrary to the synergetic effect on multiplesclerosis by combinational OTU0001 and 2 in our electrochemical analysis, the mixture ofequal cell densities of OTU0002 and OTU0001 in the EC reactor showed a decreased currentproduction (~35 nA/cm2, Figure S4) compared to OTU0002 alone current production,implicating a non-synergetic dependency with each other and absence of Quorum sensing(QS) mediated communication, thus electron transfer reduced to half. However, these dataclearly indicate the impact of OTU0001 on OTU0002 either by diminishing its activity orsharing the metabolic electrons. The decrease could be due to OTU0001’s antimicrobialactivity as L. reuteri, which is similar, produces antimicrobial molecules that inhibit harmfulmicrobes, and change the host’s microbiota composition [33].Given, electron mediators affect interactions with the bacterial surface, eventually,the transfer of electrons can be enhanced to maintain redox homeostasis within cells. [34].The electron transfer of OTU0002 associated with metabolism may work together to en-hance pathogenicity and the development of biofilms. Considering the ability of cell-surfaceredox regents to facilitate lateral electron transport over microbial aggregation [35], thepolymicrobial biofilm containing this electrogenic gut pathogen may serve as a syner-gistic vehicle for the progression of the disease’s symptoms through electron exchange.Furthermore, EET linked to population-level phenotypes can be crucial during microbialcolonization and biofilm formation. This phenomenon allows microorganisms to coor-dinate their behaviour and adapt to environmental changes. It is also possible that thepure culture biofilm of the electrogenic pathogen has electrical conductivity. In environ-mental EET-capable bacteria, such as Geobacter, clonal biofilm has electric conductivityover distances on the centimetre scale [36] due to membrane-bound cytochromes or redoxshuttlers in biofilms. Thus, OTU0002 may have the long-range electron transfer capabilityas proposed in Figure S5. Pathogen EET could be a potential target for the development ofnew antimicrobial strategies that disrupt the electron transfer pathway and prevent biofilmformation. Based on the non-electrochemical activity of OTU0001, which is a probiotichealthy commensal [33], combined with the absence of redox species in both DPV and TEManalyses, it supports the bias of EET capability toward pathogenicity.3.3. Phylogenetic Relevance of OTU0002 with Other EET-Capable PathogensWe analysed the 16S rRNA sequences of this gut pathogen to determine its phy-logenetic relevance with previously reported gut pathogens capable of EET. Using theNational Center for Biotechnology Information (NCBI) database, we performed sequencealignment and compared the ribosomal RNA gene sequences of OTU0002 with represen-tative microbial community sequences from known EET-capable gut microbes such asListeria monocytogenes [14], Enterococcus faecalis [17], and Faecalibacterium prausnitzii [37].Additionally, we overlaid the electrochemically enriched EET-capable gut pathogens En-terococcus avium and Klebsiella pneumoniae [19] as illustrated in Figure 5. Interestingly, theanalysis revealed that OTU0002 exhibited a close relationship with Listeria, a Gram-positivefoodborne human gut pathogen. Listeria is known to possess an EET mechanism that iscoupled with a soluble electron acceptor, flavin molecules along with a membrane-boundprotein complex involved in its EET mechanism. Given flavin increased current production,the flavin-cofactor enzyme is a potential EET mechanism in OTU0002 because flavin-boundcell surface enzymes mediate EET in some model microbes [38]. While the physiologicalrole of EET in Listeria monocytogenes has not been clarified, the mutant strains lacking thegene encoding EET-flavoenzymes are responsible for mouse model infection [14]. Upon theidentification of the critical genes for EET in OTU0002, their impact on biofilm formationand multiple sclerosis, therefore, should be studied in mouse models.Microorganisms 2024, 12, 257 9 of 11Microorganisms 2024, 12, x FOR PEER REVIEW  10  of  13    Figure 5. Phylogenetic tree depicting the identity of EET capable gut pathogens and the OTU0002, based on 16S rRNA sequence alignment, generated using MUSCLE; the neighbour-joining method was employed for phylogenetic tree construction (Scale bar: 0.20 substitutions per site). 4. Conclusions Our analysis using electrochemistry reveals that OTU0002, a human pathogen that is directly linked to multiple sclerosis, has the ability to transfer electrons in a manner that is dependent on cell density and active metabolism. In contrast, OTU0001, which coexists with OTU0002 in the gut of mice with autoimmune diseases, did not produce significant electrical current under the same electrochemical conditions. It is possible that OTU0001 might benefit from receiving electrons from OTU0002 for IET within the biofilm leading to  the progression of disease. The use of redox mediators such as flavins suggests  that these pathogens might use the available redox shuttles in the gut niche to progress their EET. Studying the electron transfer mechanism and electron uptake capabilities of both strains could be a valuable line of future research. Such research would not only enhance our understanding of microbial electron transfer processes but also broaden our perspec-tive on microbial interactions in diverse environmental and biological settings. Supplementary  Materials:  The  following  supporting  information  can  be  downloaded  at: www.mdpi.com/xxx/s1. Figure S1: An illustration showing the experimental procedures that were used  in  this  study  (A) Preculture  growth medium  and  bottle  condition used  for OTU0001  and OTU0002 growth, which were harvested, washed in defined medium (DM), and used for electro-chemical  experiments. Similar  cells were  subjected  to 3,3ʹ-diaminobenzidine  (DAB)  staining  fol-lowed by TEM observations as presented  in Figure 2.  (B) Electrochemical setup containing  three electrodes, indium tin-doped oxide (ITO) electrode (surface area: 3.14 cm2) poised at +0.4 V (versus SHE) as working electrode (W.E.), platinum wire as counter electrode (C.E.) and Ag/AgCl (saturated KCL) as reference electrode (R.E.). Separate electrochemical setups were used to obtain the Figures 1, 3 and 4 data. More than two repeated experimental analyses were conducted and representative data was shown. Figure S2: Medium swapping analysis of OTU0002 in the electrochemical reactor. A) Supernatant replacement during the current production of OTU0002 at +0.4 V (versus SHE). At the indicated times, the medium was removed and replaced with a fresh defined medium contain-ing 10 mM glucose (blue line) or cell-free spent medium (red line), leading to a decrease in current production. The same tendency was confirmed in two individual experiments. B) Differential pulse voltammograms in the presence of OTU0002 before (black line), after fresh medium exchange (blue line), and DPV of biofilm after spent medium exchange  (red dotted  line). Figure S3: Correlation between cell density  (OD600nm) and maximum current produced  (µA) by OTU0002  taken  from Figure 5. Phylogenetic tree depicting the identity of EET capable gut pathogens and the OTU0002,based on 16S rRNA sequence alignment, generated using MUSCLE; the neighbour-joining methodwas employed for phylogenetic tree construction (Scale bar: 0.20 substitutions per site).4. ConclusionsOur analysis using electrochemistry reveals that OTU0002, a human pathogen that isdirectly linked to multiple sclerosis, has the ability to transfer electrons in a manner that isdependent on cell density and active metabolism. In contrast, OTU0001, which coexistswith OTU0002 in the gut of mice with autoimmune diseases, did not produce significantelectrical current under the same electrochemical conditions. It is possible that OTU0001might benefit from receiving electrons from OTU0002 for IET within the biofilm leading tothe progression of disease. The use of redox mediators such as flavins suggests that thesepathogens might use the available redox shuttles in the gut niche to progress their EET.Studying the electron transfer mechanism and electron uptake capabilities of both strainscould be a valuable line of future research. Such research would not only enhance ourunderstanding of microbial electron transfer processes but also broaden our perspective onmicrobial interactions in diverse environmental and biological settings.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms12020257/s1. Figure S1: An illustration showing theexperimental procedures that were used in this study (A) Preculture growth medium and bottle conditionused for OTU0001 and OTU0002 growth, which were harvested, washed in defined medium (DM),and used for electrochemical experiments. Similar cells were subjected to 3,3’-diaminobenzidine (DAB)staining followed by TEM observations as presented in Figure 2. (B) Electrochemical setup containingthree electrodes, indium tin-doped oxide (ITO) electrode (surface area: 3.14 cm2) poised at +0.4 V (versusSHE) as working electrode (W.E.), platinum wire as counter electrode (C.E.) and Ag/AgCl (saturatedKCL) as reference electrode (R.E.). Separate electrochemical setups were used to obtain the Figures 1, 3and 4 data. More than two repeated experimental analyses were conducted and representative data wasshown. Figure S2: Medium swapping analysis of OTU0002 in the electrochemical reactor. A) Supernatantreplacement during the current production of OTU0002 at +0.4 V (versus SHE). At the indicated times, themedium was removed and replaced with a fresh defined medium containing 10 mM glucose (blue line)or cell-free spent medium (red line), leading to a decrease in current production. The same tendencywas confirmed in two individual experiments. B) Differential pulse voltammograms in the presenceof OTU0002 before (black line), after fresh medium exchange (blue line), and DPV of biofilm afterspent medium exchange (red dotted line). Figure S3: Correlation between cell density (OD600nm) andmaximum current produced (µA) by OTU0002 taken from Figure 2A Time vs Current profile of differenthttps://www.mdpi.com/article/10.3390/microorganisms12020257/s1https://www.mdpi.com/article/10.3390/microorganisms12020257/s1Microorganisms 2024, 12, 257 10 of 11cell densities 0.1, 0.2, 0.5, and 1.0, showing the non-linearity. Figure S4: Current vs time measurements ofmixed culture of OTU0001 and OTU0002, conducted in anaerobic reactors equipped with ITO electrodes(surface area: 3.14 cm2) poised at +0.2 V vs Ag/AgCl in the presence of 10 mM glucose and the OD600 of0.25 of each strain initial cell density added to the reactors. The arrow position indicates the time of celladdition in the electrochemical reactor. Figure S5: Schematic of possible long-range electron transportmechanism by OTU0002 cells. At high OD600 the EET is enhanced by the concentration of cell-releasedredox active product, which acts as an electron mediator. The cell-released mediator attaches to the celland transfers the electrons to the electrode via reduction and oxidation state cycling.Author Contributions: Conceptualization, D.N., W.M. and A.O.; methodology, D.N. and W.M.;validation, D.N., W.M. and A.O.; formal analysis, W.M., D.N. and A.O.; investigation, D.N. andW.M.; resources, A.O.; data curation, W.M. and D.N.; writing—original draft preparation, D.N. andW.M.; writing—review and editing, A.O.; supervision, A.O.; project administration, A.O.; fundingacquisition, A.O. All authors have read and agreed to the published version of the manuscript.Funding: The financial support for this work was provided by a Grant-in-Aid for Research fromthe Japan Society for Promotion of Science KAKENHI (Grant Nos. 20H05590 and 22H02265)and PRIME, the Japan Agency for Medical Research and Development (19gm6010002h0004 and22ae0121044h0002). This work was also supported by JST, PRESTO Grant Number JPMJPR19H1, andCREST Grant Number JPMJCR19H4.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Data are contained within the article or Supplementary Materials.Acknowledgments: We thank Eiji Miyauchi and Hiroshi Ohno of RIKEN Center for IntegrativeMedical Sciences, Yokohama, Japan, for kindly providing the OTU0001 and OTU0002 strains.Conflicts of Interest: The authors declare no conflict of interest.References1. Nealson, K.H.; Little, B. Breathing manganese and iron: Solid-state respiration. Adv. Appl. 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MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.https://doi.org/10.1016/j.isci.2021.102068https://www.ncbi.nlm.nih.gov/pubmed/33554070https://doi.org/10.1099/13500872-141-8-1985https://www.ncbi.nlm.nih.gov/pubmed/7551061https://doi.org/10.1128/mBio.00626-17https://www.ncbi.nlm.nih.gov/pubmed/29636430https://doi.org/10.1021/acs.biochem.8b00600https://doi.org/10.3389/fmicb.2018.03267https://doi.org/10.1016/j.cell.2020.07.006https://doi.org/10.1073/pnas.1522149113https://doi.org/10.1073/pnas.1117592109https://doi.org/10.1038/nature15512https://www.ncbi.nlm.nih.gov/pubmed/26375009https://doi.org/10.1128/AEM.03837-12https://www.ncbi.nlm.nih.gov/pubmed/23377933https://doi.org/10.1038/s41586-020-2634-9https://doi.org/10.1128/AEM.72.5.3236-3244.2006https://www.ncbi.nlm.nih.gov/pubmed/16672462https://doi.org/10.1021/ac50008a031https://doi.org/10.1016/S0065-1281(82)80023-8https://doi.org/10.1038/ismej.2015.181https://doi.org/10.1021/acs.est.3c00601https://doi.org/10.1002/elps.201400451https://doi.org/10.1093/ajcn/77.6.1352https://doi.org/10.3389/fmicb.2018.00757https://www.ncbi.nlm.nih.gov/pubmed/29725324https://doi.org/10.1038/s41598-017-07593-yhttps://www.ncbi.nlm.nih.gov/pubmed/28798305https://doi.org/10.3389/fenrg.2019.00087https://doi.org/10.1038/nnano.2011.119https://www.ncbi.nlm.nih.gov/pubmed/21822253https://doi.org/10.1038/ismej.2012.5https://www.ncbi.nlm.nih.gov/pubmed/22357539https://doi.org/10.1002/celc.201402151 Introduction  Materials and Methods  Cell Culture Preparation  Transmission Electron Microscopy  Whole-Cell Electrochemical Analysis of Pure Strains  Supernatant Exchange Experiments during the Current Producing Condition  Scanning Electron Microscopy (SEM)  Results and Discussion  Electrochemical Characterization of OTU0001 and OTU0002  Potential Involvement of Exogenous and Endogenous Redox Mediators on the Microbial Current Production of OTU0002  Phylogenetic Relevance of OTU0002 with Other EET-Capable Pathogens  Conclusions  References