Integrity of the Escherichia coli O157:H7 cell wall and membranes after chlorine dioxide treatment

Authors: Bridges, David; Lacombe, Alison; Wu, Vivian

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2020
Publication Date: 5/15/2020
Citation: Bridges, David F., Alison Lacombe, and Vivian C. H. Wu. (2020). "Integrity of the Escherichia coli O157:H7 Cell Wall and Membranes after Chlorine Dioxide Treatment". Frontiers in Microbiology 11:888. doi: 10.3389/fmicb.2020.00888
DOI: https://doi.org/10.3389/fmicb.2020.00888

Interpretive Summary: The use of chlorine dioxide (ClO2) as an antimicrobial has increased in popularity over the past decade. The ability of ClO2 to kill bacteria has been thoroughly reported but, how ClO2 harms bacteria is still poorly understood. Therefore, the objective of this study was to observe changes and damages to the bacterial cell wall using Escherichia coli O157:H7 as a model pathogenic organism. Utilizing a mixed-method approach which implemented both quantitative and qualitative methodologies, the cell wall of E. coli after ClO2 treatment was demonstrated to be not significantly damaged. In comparison, equivalent concentrations of sodium hypochlorite (NaOCl) resulted negligible levels of reduction while causing observable changes to the cell wall. It is commonly assumed that oxidizing antimicrobials (e.g. NaOCl, ClO2, and O3) all kill bacteria by similar methods of oxidative damage. However, the results of this study imply that ClO2 damages E. coli significantly different than what would be expected by treatment with an oxidizer.

Technical Abstract: 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 (ClO2) treatment but information regarding how ClO2 exposure affects bacteria is limited. In the present study, a mixed-method approach utilizing both quantitative and qualitative methodologies was used to observe and quantify Escherichia coli O157:H7 membrane damage after exposure to aqueous ClO2. E. coli O157:H7 was exposed to ClO2 (2.5, 5, or 10 ppm) for 5, 10, or 15 min. For comparison, controls of 0.1% peptone, 70% isopropanol, and 10 ppm NaOCl were applied for 15 min. After treatment, cells were enumerated on selective media and simultaneously analyzed for cellular 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). Fluorescent emissions from each probe were expressed as relative fluorescent units (RFU) and compared to controls. Bacterial log reductions after ClO2 treatment ranged from 0.17-5.47. Changes in RFU after the LIVE/DEAD (membrane permeability) and 2-NBDG (glucose uptake) assays were not consistent with viability, indicating there could be alterations in membrane permeability and metabolism. Depolarization (DiBAC4(3)) was observed after NaOCl treatment, however, cells treated with ClO2 showed results like those treated with water (P<0.05). Accumulation of MDA (lipid peroxidation) was detected only after 10 ppm ClO2 treatments, indicating that membrane peroxidation occurred at higher concentrations. Images takes using transmission electron microscopy (TEM) revieled that separation of the cell wall from the cytosol occurred after the 10-ppm ClO2 treatment, but the cell wall itself appeared to be unbroken. These data suggest that ClO2 damage to E. coli O157:H7 is not primarily located at the cell wall and harms cells significantly different than NaOCl at comparable concentrations.