Metabolic Current Production by an Oral Biofilm Pathogen Corynebacterium matruchotii

Abstract

<jats:p>The development of a simple and direct assay for quantifying microbial metabolic activity is important for identifying antibiotic drugs. Current production capabilities of environmental bacteria via the process called extracellular electron transport (EET) from the cell interior to the exterior is well investigated in mineral-reducing bacteria and have been used for various energy and environmental applications. Recently, the capability of human pathogens for producing current has been identified in different human niches, which was suggested to be applicable for drug assessment, because the current production of a few strains correlated with metabolic activity. Herein, we report another strain, a highly abundant pathogen in human oral polymicrobial biofilm, Corynebacterium matruchotii, to have the current production capability associated with its metabolic activity. It showed the current production of 50 nA/cm2 at OD600 of 0.1 with the working electrode poised at +0.4 V vs. a standard hydrogen electrode in a three-electrode system. The addition of antibiotics that suppress the microbial metabolic activity showed a significant current decrease (&gt;90%), establishing that current production reflected the cellular activity in this pathogen. Further, the metabolic fixation of atomically labeled 13C (31.68% ± 2.26%) and 15N (19.69% ± 1.41%) confirmed by high-resolution mass spectrometry indicated that C. matruchotii cells were metabolically active on the electrode surface. The identified electrochemical activity of C. matruchotii shows that this can be a simple and effective test for evaluating the impact of antibacterial compounds, and such a method might be applicable to the polymicrobial oral biofilm on electrode surfaces, given four other oral pathogens have already been shown the current production capability.</jats:p>

Pathogens electrogenicity as a tool for in-situ metabolic activity monitoring and drug assessment in biofilms

“…The most facile method would be the addition of inhibitors for specific metabolic processes during SA. For example, the addition of ampicillin inhibits cell wall formation and ultimately affects cell growth, and triclosan, a glycolysis inhibitor, at lethal concentrations results in a significant decrease in the growth of S. mutans, and C. matruchotii (Naradasu et al, 2020a(Naradasu et al, , 2020c (Table 1). It is important to mention that only lethal concentrations have been tested for antibiotic dosage in pathogen's current-producing reactors, and more detailed studies will help in establishing the dose-dependent behavior of current production.…”

“…However, for other human electrogenic pathogens such as Klebsiella pneumoniae, Enterococcus avium, Enterococcus faecalis, Capnocytophaga ochracea, A. actinomycetemcomitans, and P. gingivalis, current production/mineral reduction mechanisms are unclear because they do not have any genes that encode conclusive or well-characterized EET pathways (Naradasu et al, 2018(Naradasu et al, , 2020bPankratova et al, 2018;Zhang et al, 2020b). Recent studies have identified redox species at the cell surface and membrane in S. mutans (Naradasu et al, 2020a), C. matruchotii (Naradasu et al, 2020c), A. actinomycetemcomitans, and P. gingivalis (Naradasu et al, 2020b) by DAB staining and electrochemical analysis suggesting that EET might be involved in current production. The observed redox potential ranged from À125 mV to +250 mV, which is comparatively negative than those in the gut microbiome ( Table 1).…”

“…This oxygen gradient suggests the possibility of long-range EET in oral biofilms, where anaerobes deep in the biofilm can transfer electrons to the aerobic space for reduction by oxygen (as the final electron acceptor). The main members of the oral biofilm, Streptococcus mutans, Capnocytophaga ochracea, Corynebacterium matruchotii, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis (Naradasu et al, 2020a(Naradasu et al, , 2020b(Naradasu et al, , 2020cZhang et al, 2020b), can produce electric current with cell-surface redox species. Given cell-surface redox regents can mediate lateral electron transport across microbial aggregation (Okamoto et al, 2012;Chong et al, 2019), it is possible that these electrogenic pathogens can exchange electron in the polymicrobial biofilm.…”

“…Microscopic observations of electrogenic biofilms and single cell metabolic activity (A-C) Scanning electron micrographs showing multilayered biofilm formation by (A) S. mutans and (B) C. matruchotii, and monolayer by (C) P. gingivalis on the ITO electrode after multiple washings. Scale bar, 5 mm(Naradasu et al, 2020a(Naradasu et al, , 2020b(Naradasu et al, , 2020c. (D) NanoSIMS image of cells attached to the ITO electrode showing the 12 C 15 Nion pixel intensity for S. mutans.…”

Novel Biologically Active Molecules, Biomaterials, and Nanoparticles for Microbial Biofilm Control in Human Medicine

“…In their study, Naradasu et al demonstrated that the efficacy of antimicrobials is evidenced by the suppression of microbial metabolic activity correlated with a significant current decrease. The method described may be applicable to the polymicrobial oral biofilms on the electrode surface [ 16 ].…”