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.
Highlights
Chlorine in the form of sodium hypochlorite (NaOCl) or calcium hypochlorite [Ca(OCl2)], is the most commonly used antimicrobial for produce washes and water sanitation
Previous studies that treated E. coli, P. aeruginosa, and S. aureus with ClO2 (≤5 mg/L) achieved maximum log reductions of >5 log CFU/mL and found that after treatment there was an increase in bacterial membrane permeability
A day prior to experimentation, a loopful of E. coli O157:H7 culture maintained on tryptic soy agar (TSA) was used to inoculate 10 mL tubes of tryptic soy broth (TSB) which were incubated for 16–18 h at 37◦C
Summary
Chlorine in the form of sodium hypochlorite (NaOCl) or calcium hypochlorite [Ca(OCl2)], is the most commonly used antimicrobial for produce washes and water sanitation. ClO2 washes have been previously demonstrated to effectively reduce levels of pathogenic bacteria including, but not limited to, Shiga toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Yersinia enterocolitica on a variety of produce (Gómez-López et al, 2009; Wu, 2016; Praeger et al, 2018; Tadepalli et al, 2018; Bridges et al, 2019). Additional examination of transmission electron microscopy (TEM) micrographs further showed that there was no noticeable morphological or cell wall damage (Ofori et al, 2018, 2017)
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