INTRODUCTION

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Listeria monocytogenes is a ubiquitous, gram-positive, aerobic to facultative anaerobic bacterium. It is the causative agent of the disease listeriosis and was discovered almost 90 years ago (6, 10). In 1981, it was recognized as an important foodborne pathogen. L. monocytogenes causes a high percentage of the fatalities due to foodborne disease, exceeding even Clostridium botulinum and Salmonella. It has been suggested that listeriosis may be the leading fatal foodborne disease in the United States (8). Consumption of foods contaminated with L. monocytogenes can cause both sporadic illness and foodborne disease outbreaks. The annual rate of listeriosis incidence is 0.7 case per 100,000 persons. The rate is three times higher for persons over 70 years old and is 17 times higher for pregnant women (30). The overall fatality rate for recent outbreaks is 33% (23, 25).

Ozone (O₃) is a powerful oxidizing agent, with an oxidation potential of 2.07 V in alkaline solution, second only to fluorine (28). Dominquez et al. (5) found that ozone was more effective than chlorine in the destruction of Legionella pneumophila. Herbold et al. (14) showed that ozone inactivation of hepatitis A virus and Escherichia coli was faster at 10°C than at 20°C. Sugita et al. (31) found that ozone was highly effective in the destruction of Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida in seawater. Ozone is a protoplasm oxidant, and its bactericidal action is extremely rapid. Approximately 10 min was the critical time for all of the microorganisms tested with an O₃ concentration of 0.18 ppm. Ozone may replace chlorine as a common sanitizing agent in the food industry.

The ability of ozone to kill Salmonella typhi, Vibrio cholerae, and Bacillus anthracis was demonstrated as early as 1892. Sterilization of drinking water showed the destruction of all pathogens and saprophytic microbes encountered in water even when it was heavily contaminated (30). Whiteside and Hassan (35) demonstrated the effectiveness of ozone at causing inhibition of growth and loss of viability of E. coli. The critical dose for E. coli was between 0.40 and 0.50 ppm (32). Komanapalli and Lau (20) found that the E. coli membrane was the primary site of attack by ozone, with other cell sites subsequently damaged. This group proposed that sulfhydryl groups in the membrane were the primary targets (21). The proposed killing mechanism of ozone is as follows. Unsaturated lipids are prominent constituents of the cytoplasmic membrane. Upon exposure to ozone, the olefinic bonds are attacked to form an ozonide. This action starts the destruction of the cell's ability to function and may even be sufficient to cause the death of weaker cells. This ozonide has a high oxidation potential, is unstable, and exerts its own disinfecting action by attacking enzymes, sulfhydryl groupings, or aldehydes, releasing peroxyl compounds, which are also disinfectants. Finally, the cell is lysed and the cytoplasm is dispersed. Thus, the action of ozone is characterized by the proliferation of many other oxidizing substances which can compete with or supplement the action of ozone to destroy critical sites within the cell or to generally oxidize protoplasm. This cascade effect is unique to ozone and its decomposition products (32). Ozone has been found to cause single- and double-stranded DNA breaks in E. coli (13).

This study examined the effects of ozone on L. monocytogenes in different phases of growth and at different temperatures. We also evaluated the use of ozone on cabbage inoculated with L. monocytogenes. Another main objective of this study was to examine the role of in vivo catalase (CA) and superoxide dismutase (SOD) in protecting the cell from ozone.

	MATERIALS AND METHODS

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Bacterial strains. L. monocytogenes strains 19112 and 7644 were obtained from the American Type Culture Collection, Manassas, Va.; strains 10403S and SLCC 5764 were obtained from Daniel A. Portnoy, University of Pennsylvania, Philadelphia; strain Scott A was obtained from Larry Beuchat, University of Georgia Experimental Station, Experiment; and strain LO28 was obtained from J. Claudio Pérez-Diaz, Madrid, Spain.

Growth conditions. Frozen stocks of the cultures were prepared by inoculating 10 ml of tryptic soy broth (TSB; Fisher Scientific, Pittsburgh, Pa.) with 0.1 ml of an overnight stationary-phase inoculum. These tubes were then vortexed, frozen, and stored at 20°C. As needed, stocks were thawed and inoculated into 250-ml Erlenmeyer flasks containing 90 ml of TSB and grown at 37°C in a gyratory shaking water bath (New Brunswick Scientific, Edison, N.J.) to early stationary phase as determined by growth curves obtained by using a DU-40 spectrophotometer (Beckman, Irvine, Calif.). Mid-exponential phase was defined as an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.7; early stationary phase was defined as an OD₆₀₀ of 1.0 to 1.1, and late stationary phase was defined as an OD₆₀₀ of 1.2 to 1.3. When examining CA and SOD activities, we prepared test cultures with TSB-2.5% NaCl. This level of NaCl was added for maximum production of enzyme activities (26). These cultures were then harvested by centrifugation (16,300 × g, 10 min, 4°C) and suspended in 100 ml of sterile distilled water (dH₂O), 10 mM phosphate-buffered saline (PBS; 130 mM NaCl, pH 7.4), or 50 mM potassium phosphate buffer (PPB; pH 7.4).

Ozone apparatus. An Infinity Corona Discharge Ozone Generator (CD-7; DEL Industries, San Luis Obispo, Calif.) or a benchtop UV ozone generator (ZO-151; DEL Industries) and an ozone sensor (1054B; Rosemount Analytical, Irvine, Calif.) were used to generate and detect the levels of ozone. Residual ozone was also determined by the indigo colorimetric method (1). Ozone was diffused-bubbled in a 1-liter spinner flask with 900 ml of sterile dH₂O, PBS, or PPB to the proper ozone concentration. The suspended culture was then added to this flask, and aliquots were taken at different intervals. These aliquots were diluted in 0.1% peptone and plated onto tryptic soy agar (Fisher Scientific) and incubated at 37°C. Plates were read after 36 to 48 h. Results were reported as CFU per milliliter. Cabbage was cut into 25-g pieces, and each piece was inoculated with 1 ml of early stationary-phase cells (10⁸ cells/ml; OD₆₀₀, 1.0 to 1.1). This culture was spread aseptically over the cabbage and allowed to dry for at least 1 h. Each sample was then submerged in 1 liter of sterile dH₂O and ozonated. Heterotrophic plate counts (HPC) were determined on Plate Count Agar (Fisher Scientific), and L. monocytogenes counts were determined on McBride Listeria Agar (Fisher Scientific) containing 0.2 g of cycloheximide per liter to enhance selectivity. These plates were also incubated at 37°C for 36 to 48 h.

Enzyme activities. CA and SOD activities were determined prior to exposure to ozone in accordance with the procedure described by Dallmier and Martin (3). One unit of CA decomposed 1 µmol of H₂O₂ per min at 25°C and pH 7.0, whereas the H₂O₂ concentration fell from 10.3 to 9.2 µmol/ml of reaction mixture. SOD activity was measured by the cytochrome c reduction method of McCord and Fridovich (24). One unit of SOD activity was defined as the amount required to inhibit the rate of reduction of cytochrome c by 50%.

Statistical analyses. Statistical analyses were performed using StatView 512+ Version 1.2 (Brain Power Inc., Calabasas, Calif.). A one-way analysis of variance was used to determine any significant differences between the tested strains. The means and standard errors of the means of triplicate experiments are shown in all of the figures and tables.

	RESULTS AND DISCUSSION

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Ozonation of L. monocytogenes in dH₂O and PBS. The effects of ozone on six strains of L. monocytogenes were evaluated. Figure 1 shows the inactivation of L. monocytogenes subjected to ozone at 0.25 ppm in dH₂O at 24°C. Many studies have shown that death rate kinetics using ozone on a variety of bacteria and viruses exhibit a biphasic curve over an extended period of time (2, 16, 18, 27). This biphasic curve is also evident in Fig. 1.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Ozone (0.25 ppm) inactivation of L. monocytogenes 19112 (), Scott A (), 10403S (), SLCC 5764 (×), 7644 (*), and LO28 () in dH2O at 24°C.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Ozone inactivation of L. monocytogenes 10403S (×, 0.40 ppm; , 1.00 ppm) and SLCC 5764 (, 0.40 ppm; , 1.00 ppm) at 24°C.

Ozone exposure of different-phase cells at different temperatures. The log reductions of L. monocytogenes strains Scott A, 10403S, and SLCC 5764 after 2 min of exposure to O₃ at 0.25 ppm in PBS (24°C) are shown in Table 1. Mid-exponential-, early stationary-, and late stationary-phase cells were evaluated. Mid-exponential- and late stationary-phase cells were more sensitive to ozone than were early stationary-phase cells. However, strain Scott A showed no significant decreases between phases of growth while 10403S showed a significant decrease (P < 0.05) between the early and late stationary phases. Mid-exponential- and late stationary-phase cells were significantly more sensitive to ozone (P < 0.05) than were early stationary-phase cells of strain SLCC 5764. In general, mid-exponential- and late stationary-phase cells were more sensitive to adverse conditions than were early stationary-phase cells.

Ozone inactivation of L. monocytogenes on cabbage. The ability of ozone to kill L. monocytogenes inoculated onto cabbage is shown in Table 3. Cabbage (25-g pieces) was inoculated with 1 ml of an overnight early stationary-phase culture (OD₆₀₀, 1.0 to 1.1). L. monocytogenes counts and plate counts were examined after 2 and 5 min of exposure to ozone at 1.00 ppm in dH₂O (24°C). After 2 min, L. monocytogenes strains Scott A, 10403S, and SLCC 5764 showed 100, 75, and 70% inactivation (McBride Listeria Agar), respectively, while HPC were reduced by 18, 23, and 32%. After 5 min, all L. monocytogenes strains were inactivated while HPC were reduced by 42, 38, and 41%. Cabbage was also ozonated without the inoculation with L. monocytogenes. There were 69 and 79% decreases in HPC after 2 and 5 min of exposure, respectively. These results suggest that ozone at 1.00 ppm may be effective at inactivating all L. monocytogenes cells and in reducing the HPC.

Survival of wild-type and mutant strains. The residual ozone concentrations in sterile PPB at 4, 24, and 37°C determined prior to the addition of listerial cells were 0.30, 0.25, and 0.19 mg/liter, respectively, after 40, 60, and 90 min of exposure using the benchtop ozone generator. The time required to reach saturation was dependent on the temperature solubility of ozone (A. D. Venosa and E. J. Opatken, 52nd Annu. Pollut. Contr. Fed. Conf., Houston, Texas, 1979). The ability of ozone to kill each of the three listerial strains during the mid-exponential, early stationary, and late stationary phases when the bacteria were exposed to ozone-saturated PPB at 4, 24, and 37°C was examined. Results for all of the strains grown at the three temperatures were similar: rapid death in the first 2 to 4 min of ozone exposure, followed by a more gradual decrease in cell number. Complete inactivation was found in 6 to 20 min, depending on the strain and growth phase. All of the listerial inactivation experiments demonstrated similar biphasic death curves.

Influence of CA and SOD. Another objective of this study was to examine the effects of in vivo CA and SOD on ozone sensitivity and resistance. A wild-type strain and two mutants were utilized, i.e., L. monocytogenes strains 10403S (wild type), 1370 (CA SOD⁺), and DHL1 (CA⁺ SOD). The CA and SOD activities of the three strains are presented in Table 4. The highest CA and SOD activities were found in early stationary-phase cells of all three strains (when present). The mid-exponential-phase cells had enzymatic activities similar to those of early stationary-phase cells (P > 0.05), while the activities decreased in cells in the late stationary phase. Strain 1370 produced significantly more SOD than did strain 10403S (P < 0.05). This increased production of SOD may be in compensation for the lack of CA. An approximately twofold increase in SOD production in a CA-negative mutant compared with a CA-positive strain was previously observed when listerial cells were exposed to oxygen (12, 34). No overproduction of CA was observed in the SOD-negative mutant DHL1, and the level was similar to that of strain 10403S. Imlay and Linn (15) obtained similar results with a SOD-negative strain of E. coli compared to a SOD-positive strain.