Electrochemical Characterization of Current‐Producing Human Oral Pathogens by Whole‐Cell Electrochemistry

Abstract

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.

Metabolic Current Production by an Oral Biofilm Pathogen Corynebacterium matruchotii

“…The multicellular filaments with hundreds of cells arranged end-to-end resulted in the enhanced EET ability to electrodes [ 36 ]. Similarly, given that filamentous C. matruchotii is discovered as an electroactive oral pathogen in this study and some other oral biofilm pathogens such as Streptococcus mutans [ 16 ], Aggregatibacter actinomycetemcomitans [ 6 ], Porphyromonas gingivalis [ 6 ] and Capnocytophaga ochracea [ 38 ] have already been shown to have EET capability are well connected with C. matruchotii , which nucleates the plaque-characteristic consortium according to the spatial organization in oral biogeography [ 17 ], it can be anticipated that long range electron transport is supported by C. matruchotii and the whole oral biofilm is electrically conductive. Based on the previous findings that in the oral polymicrobial biofilm, consortium consists of radially arranged taxa, organized around cells of filamentous Corynebacteria with anaerobic taxa in the interior and facultative or obligate aerobes tend at the periphery of the consortium [ 17 ], we can purpose that this arrangement may support electrically coupled organics oxidation and oxygen reduction in oral polymicrobial biofilm as in the cases of long-range EET in natural environments.…”

“…Single-potential amperometry (SA) and differential pulse voltammetry (DPV) measurements were conducted with an automatic polarization system (VMP3, Bio Logic Company, France) as reported earlier [ 22 , 42 ]. The following conditions were used for DPV measurements: pulse increments 5.0 mV, pulse amplitude 50 mV, pulse width 300 ms and a pulse period of 5.0 s. Supernatant exchange experiments and scanning electron microscopy (SEM) observations were performed as explained in our previous study [ 6 ].…”

“…Recently, EET-capable bacteria are also identified in the human microbiome Listeria monocytogenes , where instead of c -type cytochromes, a protein-bound eight-gene locus (including ndh2 ) couples with flavins shuttle electrons to the extracellular electron acceptor [ 4 ]. Few other human pathogens with inconclusive or uncharacterized EET pathways such as Klebsiella pneumoniae , Enterococcus avium , Enterococcus faecalis , Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis have also been shown to involve in current production/minerals reduction [ 5 , 6 , 7 ]. Although many pathogens employed fermentation as the source of energy without the requirement of inorganic terminal electron acceptor, EET by pathogens likely facilitate their colonization in the host’s anaerobic environment, which can be of great significance for human health.…”

“…Thus, the amperometry may be an effective technique for antimicrobial drug selection based on pathogens’ metabolic activity correlation with the current production, and hence the drug’s effectiveness against EET capable pathogens can be assessed. Recent studies showed that Streptococcus mutans [ 16 ], Aggregatibacter actinomycetemcomitans , and Porphyromonas gingivalis [ 6 ] has the current production ability with the correlation with metabolic activity. These three strains are oral pathogens and the members of the polymicrobial biofilm.…”

Electroactivity across the cell wall of Gram-positive bacteria

“…Electroactive organisms can be found in all three domains of life, being ubiquitous in distinct environments, including lakes, soils as well as in deep-sea hydrothermal vents [11] , [12] . Recently, is has been demonstrated that these organisms are also present in the human digestive system [16] , [17] , in the mouse gut microbiome [18] , [19] , [20] and oral plaque [21] , with some of them associated with infectious diseases [22] , [23] .…”