Integrity of the Escherichia coli O157:H7 Cell Wall and Membranes After Chlorine Dioxide Treatment

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 in a variety of applications but information regarding how ClO₂ affects bacteria is limited. In the present study, a mixed-method approach utilizing both quantitative and qualitative methodologies was used to observe Escherichia coli O157:H7 membrane damage after exposure to ClO₂ (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 ClO₂ 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 ClO₂ were like those treated with water (P < 0.05). Accumulation of MDA was detected only after 10 mg/L ClO₂ 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 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.

Keywords: Escherichia coli O157:H7; antimicrobial treatments; bacterial membranes; chlorine dioxide; oxidizers.

FIGURE 1

Log reduction of E. coli O157 after 2.5, 5, or 10 mg/L ClO₂ treatment for 5, 10, or 15 min compared to control treatments (A) and linear regressions of bacterial reduction over time after ClO₂ treatment (B). Data are presented as means ± standard deviations (n = 3) and significant differences (P ≤ 0.05) in bacteria reductions observed after each treatment are represented by different letters (e.g., A–F).

FIGURE 2

2-NBDG signal after 2.5, 5, or 10 mg/L ClO₂ treatment for 5, 10, or 15 min compared to control treatments (A) and linear regressions of 2-NBDG signal over time after ClO₂ treatment (B). Data are presented as means ± standard deviations and significant differences (P ≤ 0.05) in bacteria reductions observed after each treatment are represented by different letters (e.g., A–G).

FIGURE 3

SYTO 9: PI ratio after 2.5, 5, or 10 mg/L ClO₂ treatment for 5, 10, or 15 min compared to control or ClO₂ treatment. Data are presented as means ± standard deviations (n = 3) and significant differences (P ≤ 0.05) in bacteria reductions observed after each treatment are represented by different letters (e.g., A–F).

FIGURE 4

DiBAC₄(3) uptake after 2.5, 5, or 10 mg/L ClO₂ treatment for 5, 10, or 15 min compared to control or ClO₂ treatment. Data are presented as means ± standard deviations (n = 3) and significant differences (P ≤ 0.05) in bacteria reductions observed after each treatment are represented by different letters (e.g., A–E).

FIGURE 5

Transmission electron micrographs of E. coli O157 after treatment with distilled water (A), 70% isopropanol (B), 10 mg/L NaOCl (C), 2.5 mg/L ClO₂ (D), 5 mg/L ClO₂ (E), or 10 mg/L ClO₂ (F) for 15 min.

FIGURE 6

Polarization and integrity schematic of the E. coli cytoplasmic membrane during normal conditions (A) and after ClO₂ treatment if damage is localized at membrane proteins and lipids (B) or if ClO₂ damage is localized intracellularly (C).