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 (ClO 2) treatment in a variety of applications but information regarding how ClO 2 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 ClO 2 (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 2 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 2 were like those treated with water (P < 0.05). Accumulation of MDA was detected only after 10 mg/L ClO 2 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 2 treatment, but the cell wall itself appeared to be unbroken. These data suggest that ClO 2 damage to E. coli O157:H7 is not primarily located at the cell wall and harms cells significantly different than NaOCl at comparable concentrations.

Fresh Produce Safety and Quality: Chlorine Dioxide’s Role

“…Furthermore, loss of cell activity or cell death in ClO 2 -treated Pseudomonas aeruginosa and Staphylococcus aureus is correlated with the increased permeability of inner and outer cell membranes and the subsequent release of vital nuclear materials ( Ofori et al, 2018 ). At higher concentrations, ClO 2 induces accumulation of malondialdehyde (MDA) content, indicating the occurrence of membrane peroxidation ( Bridges et al, 2020 ). However, previous studies based on transmission electron microscopy did not reveal significant morphological damage or cell lysis ( Ofori et al, 2018 ; Bridges et al, 2020 ).…”

Effect of disinfection agents and quantification of potentially viable Leptospira in fresh water samples using a highly sensitive integrity-qPCR assay

“…However, the use of PMAqPCR shows that leptospiral membranes would not be directly damaged by free chlorine. Several studies have shown that free chlorine inactivates E. coli without damaging its cell membranes [49][50][51][52] further indicating that the use of PMAqPCR is not appropriate to determine the capacity of chlorine to kill bacteria.…”

Effectiveness of Aqueous Chlorine Dioxide in Minimizing Food Safety Risk Associated with Salmonella, E. coli O157:H7, and Listeria monocytogenes on Sweet Potatoes

“…In addition, the chlorine treatment can potentially produce carcinogenic compounds, such as trichloramines, as byproducts of the treatment posing public health risks [17,18]. Chlorine dioxide (ClO 2 ) has gained popularity within the last couple of decades to disinfect drinking water and wastewater, and to sanitize fresh produce and poultry [19]. ClO 2 has 2.5 times the oxidizing capability as Cl 2 and may be an effective alternative produce sanitizer [20].…”

“…Previous studies have demonstrated the efficacy of ClO 2 in reducing human pathogens including Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Yersinia enterocolitica on a variety of produce [19]. The microbial death is believed to be due to oxidation of sulfhydryl groups on cell-surface proteins and increased permeability of the outer membrane [23].…”

“…The microbial death is believed to be due to oxidation of sulfhydryl groups on cell-surface proteins and increased permeability of the outer membrane [23]. Another mechanism is its influence on internal components of the cells such as proteins and nucleic acids as well as interfering protein synthesis [19,24]. At 5 ppm, aqueous ClO 2 has demonstrated cross-contamination control of Salmonella, Escherichia coli O157:H7, and Erwinia carotovora in washing produce [16,25].…”