Implementation of Fluorescent Assays to Measure Membrane Damage to Escherichia coli O157:H7 after Exposure to Chlorine Dioxide

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/19/2019
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Introduction: Aqueous chlorine dioxide (ClO2) has demonstrated antimicrobial capabilities against foodborne pathogens and maybe a valuable alternative to bleach in an industrial setting. Purpose: This study implements fluorescence-based assays to measure membrane injury to Escherichia coli O157:H7 after exposure to aqueous ClO2. Methods: E. coli O157 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 with the following fluorescent probes for cellular damage 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. Results: Log reductions after ClO2 treatment ranged from 0.17-5.47. Reductions of 0.01, 7.87, and 0.14 were achieved after treatment with DI water, isopropanol, and NaOCl, respectively. Changes in RFU after the LIVE/DEAD and 2-NBDG assays were not correlated (Pearson’s Correlation P<0.05) with reduction as exposure to each concentration increased. Depolarization (DiBaC4(3)) was observed after NaOCl treatment, however, cells treated with ClO2 showed results like those treated with peptone water (P<0.05). Accumulation of MDA was detected after 10 ppm ClO2 treatments, indicating that membrane oxidation occurs at higher concentrations, but not at lower ones. This suggests that ClO2 damage to E. coli O157 is significantly different from NaOCl at comparable concentrations, and the damage could be dose dependent and is not correlated with viability. Significance: This study demonstrates that oxidative damage caused by ClO2 is significantly different than the damage caused by NaOCl. Understanding the impact of ClO2 on microbial physiology is important to determine appropriate post-harvest treatment conditions. Technical Interpretation: Treatments with chlorine dioxide (ClO2) as a post-harvest antimicrobial are becoming more popular due to its powerful antimicrobial properties. However, little is known about the cellular mechanism behind ClO2 damage to bacterial cells. Fluorescence-based viability assays demonstrated that ClO2 damage to bacterial damage is not correlated with viability and is significantly different than other oxidative treatments (e.g. NaOCl). Additionally, the type of cellular damage that occurred appeared to be different depending on the dose used.