Bridges David F, Lacombe Alison, Wu Vivian C H
Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA, United States.
Front Microbiol. 2020 May 15;11:888. doi: 10.3389/fmicb.2020.00888. eCollection 2020.
Treatments of wastewater and fresh produce commonly employ chlorine as an antimicrobial. However, there are increasing levels of concerns regarding the safety and antimicrobial efficacy of chlorine treatments. Numerous studies have reported the antimicrobial properties of chlorine dioxide (ClO) treatment in a variety of applications but information regarding how ClO affects bacteria is limited. In the present study, a mixed-method approach utilizing both quantitative and qualitative methodologies was used to observe O157:H7 membrane damage after exposure to ClO (2.5, 5, or 10 mg/L) for 5, 10, or 15 min. For comparison, controls of 0.1% peptone, 70% isopropanol, and 10 mg/L NaOCl were applied for 15 min. After treatment, cells were enumerated on selective media overlaid with non-selective media and simultaneously analyzed for damage using the following fluorescent probes (1) Bis-(1,3-Dibutylbarbituric Acid) trimethine oxonol (DiBAC4(3)) for membrane polarization, (2) SYTO 9/propidium iodide (LIVE/DEAD) for membrane permeability, (3) 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) for active glucose uptake, and (4) lipid peroxidation through accumulation of malondialdehyde (MDA). Bacterial log reductions after ClO treatment ranged from 0.2 to 5.5 and changes in relative fluorescence units after membrane permeability and glucose uptake assays were not consistent with viability, indicating membrane permeability and metabolism were not substantially altered. Depolarization was observed after NaOCl treatment, however, the polarity of cells treated with ClO were like those treated with water ( < 0.05). Accumulation of MDA was detected only after 10 mg/L ClO treatments, indicating that membrane peroxidation occurred at higher concentrations. Transmission electron microscopy imaging revealed that separation of the cell wall from the cytosol occurred after the 10 mg/L ClO treatment, but the cell wall itself appeared to be unbroken. These data suggest that ClO damage to O157:H7 is not primarily located at the cell wall and harms cells significantly different than NaOCl at comparable concentrations.
废水和新鲜农产品的处理通常使用氯作为抗菌剂。然而，人们对氯处理的安全性和抗菌效果的担忧日益增加。许多研究报告了二氧化氯（ClO）处理在各种应用中的抗菌特性，但关于ClO如何影响细菌的信息有限。在本研究中，采用定量和定性方法相结合的混合方法，观察O157:H7在暴露于2.5、5或10 mg/L的ClO中5、10或15分钟后的膜损伤情况。作为比较，分别应用0.1%蛋白胨、70%异丙醇和10 mg/L次氯酸钠作为对照处理15分钟。处理后，将细胞接种在覆盖有非选择性培养基的选择性培养基上进行计数，并同时使用以下荧光探针分析损伤情况：（1）双（1,3 - 二丁基巴比妥酸）三甲川草酚（DiBAC4(3)）用于膜极化；（2）SYTO 9/碘化丙啶（死活染色）用于膜通透性；（3）2 - （N - （7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂环丁烷 - 4 - 基）氨基） - 2 - 脱氧葡萄糖（2 - NBDG）用于活性葡萄糖摄取；（4）通过丙二醛（MDA）积累检测脂质过氧化。ClO处理后细菌的对数减少范围为0.2至5.5，膜通透性和葡萄糖摄取试验后相对荧光单位的变化与活力不一致，表明膜通透性和代谢没有实质性改变。次氯酸钠处理后观察到去极化，但用ClO处理的细胞的极性与用水处理的细胞相似（<0.05）。仅在10 mg/L ClO处理后检测到MDA积累，表明在较高浓度下发生了膜过氧化。透射电子显微镜成像显示，10 mg/L ClO处理后细胞壁与细胞质发生分离，但细胞壁本身似乎未破裂。这些数据表明，ClO对O157:H7的损伤主要不位于细胞壁，且在可比浓度下对细胞的损害与次氯酸钠显著不同。
