Microorganisms are known to exhibit extracellular electron transfer (EET) in a wide variety of habitats. However, as for the human microbiome which significantly impacts our health, the role and importance of EET has not been widely investigated. In this study, we enriched and isolated the EET-capable bacteria from human gut microbes using an electrochemical enrichment method and examined whether the isolates couple EET with anaerobic respiration or fermentation. Upon the use of energy-rich or minimum media (with acetate or lactate) for electrochemical enrichment with the human gut sample at an electrode potential of +0.4 V [vs. the standard hydrogen electrode (SHE)], both culture conditions showed significant current production. However, EET-capable pure strains were enriched specifically with minimum media, and subsequent incubation using the δ-MnO2-agar plate with lactate or acetate led to the isolation of two EET-capable microbial strains, Gut-S1 and Gut-S2, having 99% of 16S rRNA gene sequence identity with Enterococcus avium (E. avium) and Klebsiella pneumoniae (K. pneumoniae), respectively. While the enrichment involved anaerobic respiration with acetate and lactate, further electrochemistry with E. avium and K. pneumoniae revealed that the glucose fermentation was also coupled with EET. These results indicate that EET couples not only with anaerobic respiration as found in environmental bacteria, but also with fermentation in the human gut.
Extracellular electron transfer (EET) via cell-bound redox enzymes and/or redox shuttles is extensively studied in environmental bacteria. Meanwhile, EET capable pathogens have been identified in the human gut. However, other EET-capable bacterial niches where possible biofilm infections are prominent have scarcely been explored. Herein, we electrochemically characterized human oral biofilm pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, to examine their EET capability. Both strains showed current production with the electrode poised at + 0.4 V vs. a standard hydrogen electrode, which was associated with a decrease in electrondonor concentration, coupled with the appearance of oxidative peaks in differential pulse (DP) and cyclic voltammograms (CV).Addition of antibiotics that suppress the biosynthesis of membrane or protein showed a significant current decrease, demonstrating that current production reflects the cellular activity in these pathogens. DPV-and CV-based kinetic analyses supported by transmission electron microscopy of the cells stained for transition metals suggest a potential EET mechanism associated with the presence of redox enzymes on the cell membrane. These results could be the basis to reevaluate human oral pathogens from an electroactive point of view. The identified electrochemical activity of the two strains can be an effective test for assessing the impact of antibacterial compounds on the pathogen cellular activity on an electrode. cell secreted metabolites of the preculture medium ( Figure S1). Cell cultures were handled in COY anaerobic chamber filled with 100 % N 2 . DM was prepared according to previously described methods [32] with some modification. DM contained (per liter) the following Figure 3. Transmission electron microscopy (TEM) of 3,3'-diaminobenzidene (DAB)-stained cells of Aggregatibacter actinomycetemcomitans (AA) and Porphyromonas gingivalis (PG). Positive DAB staining with the addition of H 2 O 2 (a, c), and negative DAB staining in the absence of H 2 O 2 (b, d). Scale bars, 50 nm. DAB-stained layer thickness (e) and absorption (f) of A. actinomycetemcomitans and P. gingivalis measured from (n = 10) line profiles of TEM images under DAB-positive and -negative conditions. ChemElectroChemArticles A stock solution of Sodium Ampicillin 1.86 g/L was prepared, and the stock solution was added into the electrochemical reactor to a final concentration about 1 mg/mL. This is higher than the minimum inhibitory concentration and minimum bactericidal concentration for both planktonic and electrode-attached cells. [27] A stock solution of 50 mg/mL Kanamycin (Sigma-Aldrich) was added to a final concentration of 1 mg/mL.
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