International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan; Department of Advanced Interdisciplinary Studies, RCAST, Graduate School of Engineering, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan.
Biosens Bioelectron. 2020 Aug 15;162:112236. doi: 10.1016/j.bios.2020.112236. Epub 2020 Apr 27.
Once pathogens form a biofilm, they become more tolerant to drugs and quicker to recover from physical removal than planktonic cells. Because such robustness of a biofilm is associated with the active metabolism of its constituent microbes, establishment of a direct assay quantifying biofilm's metabolic activity is important for developing antibiofilm substrates and techniques. Current production capability via extracellular electron transport (EET) was recently found in Gram-positive pathogens, which we hypothesized to correlate with the metabolic activity of their biofilm. Here, we identified current production from the biofilm of oral pathogen Streptococcus mutans that enables the electrochemical assessments of their metabolic activity in situ which conventionally require gene insertion for a fluorescent protein expression. Single-potential amperometry (SA) showed that S. mutans produced an anodic current and formed a biofilm within 8 h on a +0.4 V electrode vs a standard hydrogen electrode (SHE) in the presence of the electron donor glucose. Current production was significantly decreased by the addition of a metabolic inhibitor Triclosan. Furthermore, the anabolic activity of a single cell using high-resolution mass spectroscopy revealed that higher current production resulted in a higher metabolic fixation of an atomically labeled nitrogen N. These results demonstrate that current production in S. mutans reflects its metabolic activity. Given electrochemical impedance spectroscopy (EIS) helps quantifying the bacterial cell adhesion on the electrode, combination of EIS and SA could be a novel assay for EET capable pathogens for quantifying their time-dependent metabolic activity, cellular electrode coverage and physiological response to antibiofilm compounds.
一旦病原体形成生物膜，它们对药物的耐受性就会增强，从物理去除中恢复的速度也会比浮游细胞更快。由于生物膜的这种稳健性与组成微生物的活跃代谢有关，因此建立直接测定生物膜代谢活性的方法对于开发抗生物膜底物和技术非常重要。最近在革兰氏阳性病原体中发现了通过细胞外电子传递（EET）进行的当前生产能力，我们假设这与它们生物膜的代谢活性有关。在这里，我们确定了口腔病原体变形链球菌生物膜中的当前生产能力，这使得能够对其代谢活性进行原位电化学评估，而传统上需要插入基因表达荧光蛋白来进行评估。单电位安培法（SA）表明，在电子供体葡萄糖存在的情况下，S. mutans 在+0.4 V 电极相对于标准氢电极（SHE）在 8 小时内产生阳极电流并形成生物膜。加入代谢抑制剂三氯生后，电流产生显著减少。此外，使用高分辨率质谱对单个细胞的合成活性进行分析表明，更高的电流产生导致更高的代谢固定原子标记氮 N。这些结果表明，S. mutans 的电流产生反映了其代谢活性。鉴于电化学阻抗谱（EIS）有助于量化细菌在电极上的粘附，EIS 和 SA 的组合可以成为一种新型的用于量化具有 EET 能力的病原体的代谢活性、细胞电极覆盖率和对抗生物膜化合物的生理反应的检测方法。
