Beverages, U.S. Food and Drug Administration, 200 C Street S.W., Washington, D.C , USA

Transcription

1 305 Journal of Food Protection, Vol. 64, No. 3, 2001, Pages Copyright q, International Association for Food Protection Survival and Growth of Escherichia coli O157:H7 Inoculated onto Cut Lettuce Before or After Heating in Chlorinated Water, Followed by Storage at 5 or 15 C YUE LI, 1 ROBERT E. BRACKETT, 1,2 JINRU CHEN, 1 AND LARRY R. BEUCHAT 1 * 1 Center for Food Safety, University of Georgia, 1109 Experiment Street, Grif n, Georgia ; and 2 Of ce of Plant and Dairy Foods and Beverages, U.S. Food and Drug Administration, 200 C Street S.W., Washington, D.C , USA MS : Received 3 August 2000/Accepted 8 October 2000 ABSTRACT This study determined the effects of mild heat and chlorine treatment followed by storage for up to 18 days at 58C or 7 days at 158C on the survival and growth of Escherichia coli O157:H7 inoculated onto fresh-cut iceberg lettuce. The ef cacy of treatment with 20 ppm chlorine in killing the pathogen on lettuce at 508C was determined. Treatment of lettuce with 20 ppm chlorine at either 20 or 508C did not result in signi cantly greater reductions in populations of E. coli O157:H7 compared to respective treatments in water without chlorine. The pathogen steadily decreased in viability on treated lettuce throughout subsequent storage at 58C for 18 days. The population increased by 2.3 to 3.2 log 10 CFU/g within 2 days, then continued to increase at a slower rate through 7 days of storage at 158C. At 4 and 7 days, signi cantly (a ) higher populations were reached on lettuce that had been treated at 508C, compared to respective samples that had been treated at 208C, regardless of the presence of 20 ppm chlorine in the treatment water. Treatment of lettuce with 20 ppm chlorine at 50 or 208C before or after inoculation with E. coli O157:H7 did not have a marked in uence on behavior of the pathogen during subsequent storage at 5 or 158C. Several outbreaks of Escherichia coli O157:H7 infections have been documented since the pathogen was rst identi ed as causing illness associated with undercooked ground beef (25). The infectious dose of E. coli O157:H7 is quite low, i.e., less than a few hundred cells (14). Most outbreaks have been linked to the consumption of foods from animal origin, although infections have also been associated with raw vegetables (5). In recent years, the frequency of outbreaks of foodborne illness associated with consumption of fresh fruits and vegetables has increased, partly as a result of an increased demand for minimally processed (ready-to-eat) produce (4, 13, 16, 23). Lettuce has been linked to outbreaks of E. coli O157:H7 infection (2, 22). Leakage of uids from lettuce tissue as a result of minimal processing may provide suf cient nutrients to support the growth of E. coli O157: H7 in the presence of high numbers of other naturally existing microorganisms (6). Cutting lettuce leaves can induce biochemical reactions that cause deterioration of sensory quality (11). Wound responses include increased phenylalanine ammonia lyase activity that initiates the biosynthesis of phenylpropanoid products (19). These phenolic compounds can be oxidized by polyphenol oxidase to form brown-colored compounds, which leads to visible discoloration along the cut edge of the lettuce leaf (21). Researchers recently reported that dipping fresh-cut lettuce in water at 45 or 558C inhibits activity of phenylalanine ammonia lyase (20) but did not describe the effects of mild heat treatment on micro ora naturally * Author for correspondence. Tel: ; Fax: ; lbeucha@cfsqe.grif n.peachnet.edu. present on the lettuce. The potential in uence of heat treatment on the behavior of human pathogens that may be present on lettuce was not addressed. Rice et al. (24) studied the sensitivity of E. coli O157: H7 to chlorine. Exposure of a cell suspension of the pathogen to 1.1 ppm free chlorine for 1 min reduced the viable population by ;4 logs. Eckert and Ogawa (15) reviewed the use of chlorinated water to wash fruits and vegetables. Signi cant reductions in populations of E. coli O157:H7 were observed after treatment of inoculated alfalfa seeds with 500 ppm chlorine (29). Spraying and soaking lettuce with chlorinated water (200 ppm) resulted in up to a 2-log reduction of E. coli O157:H7 (8), and spray treatment with 200 ppm chlorine solution or deionized water has been reported to be equally effective in killing or removing E. coli O157:H7 (6). Recently, Delaquis et al. (12) investigated the use of chlorinated water (100 ppm total chlorine) to wash shredded lettuce at 478C for 3 min. Populations of natural micro ora were reduced by 3 log 10 CFU/g. However, treatment of lettuce at an elevated temperature for the purpose of killing E. coli O157:H7 has not been reported. The study reported here was designed to determine the effects of mild heat treatment followed by storage at 5 or 158C on the survival and growth of E. coli O157:H7 inoculated onto fresh-cut iceberg lettuce. The ef cacy of treatment with 20 ppm free chlorine in killing the pathogen, as affected by temperature, was determined. MATERIALS AND METHODS Strains and preparation of E. coli O157:H7 inocula. Five strains of E. coli O157:H7 were studied: C0283 (from cattle fe-

2 306 LI ET AL. J. Food Prot., Vol. 64, No. 3 ces), EO1993 (associated with a ground beef outbreak), C7927 (associated with an apple cider outbreak), F4546 (associated with an alfalfa sprout outbreak), and H1730 (associated with a lettuce outbreak). Each strain was grown individually at 378C with vigorous shaking until reaching an A 600 of 0.5 to 1.0. Cells were harvested by centrifuging at 4,000 3 g for 15 min. Cells of each strain were transformed by electroporation (200V, 25 mf, 2.5 kv) with a green uorescent protein (GFP) plasmid (pgfpuv; ClonTech, Palo Alto, Calif.). Colonies of pgfpuv-tagged cells grown on brain heart infusion agar (BHIA; Difco, Detroit, Mich.) containing 50 mg/ml ampicillin (BHIAmp) were detected using UV (365 nm) excitation. Cells with pgfpuv were cultured via loop inocula in brain heart infusion broth containing 50 mg of ampicillin/ml (BHIAmp). Three successive 24-h transfers were made in BHIAmp prior to use as inocula. After three transfers, inocula reached a population of ;10 9 CFU/ml (A ). Preparation for chlorinated water. Distilled water containing 20 mg of available chlorine per ml (ph ) was prepared by combining water with sodium hypochlorite (Aldrich Chemical Co., Inc., Milwaukee, Wis.). A Hach test kit (model CN-66; Hach Company, Loveland, Colo.) was used to quantitate free chlorine. Preparation of cut lettuce. Iceberg lettuce (Lactuca sativa L.) was purchased from the Georgia State Farmer s Market, Forest Park, Ga. Lettuce was stored at 58C after purchase and used in experiments within 48 h. Two or three outer leaves and the core were removed and discarded. Lettuce leaves were cut into pieces using a sterile cookie cutter (6.5 cm diameter). Very thin or thick pieces were excluded. Procedures for inoculation and treatment. To mimic contamination of lettuce preceding processing and postprocessing,we inoculated lettuce before and after treatment at 50 and 208C with 20 ppm chlorine. The rst series of experiments involved inoculation before treatment. Twelve liters of 0.1 M potassium phosphate-buffered solution (PBS, ph 7.0) were combined with 50 ml of a mixture of 24-h BHIAmp cultures of 10 ml each of the ve E. coli O157:H7 strains. Pieces of cut lettuce leaves (1,500 g) were submerged with gentle agitation in the cell suspension ( C) for 1 min. Lettuce submerged in PBS for 1 min served as a control. Lettuce was placed on a wire screen lined with a nely meshed cloth to dry at C under a laminar ow hood for 1 h. Inoculated or uninoculated lettuce was then placed in a stainless-steel screen basket and submerged for 90 s in a water bath containing warm (508C) water (4 liters) with chlorine (20 ppm) or without chlorine, or water (4 liters) at 208C with chlorine (20 ppm) or without chlorine, then drained. Water was adjusted to 208C overnight. The temperature of lettuce ranged from 15 to 188C before submerging in treatment solution. Within 30 to 45 s after submerging lettuce in water at 508C, the temperature of lettuce was 47 to 508C. Lettuce was submerged in water (4 liters) at 108C for 30 s immediately after treatment in chlorinated or nonchlorinated water at 20 or 508C before drying under a laminar ow hood at C for 30 min. The second series of experiments involved inoculation of lettuce after treatment with heat and chlorine. Cut lettuce (270 g) was placed in a stainless-steel screen basket and submerged in 4 liters of water (20 or 508C) containing no chlorine or 20 ppm chlorine for 90 s, then drained as described in the rst series of experiments. Lettuce was then submerged in 4 liters of water at 108C for 30 s before combining with 6 liters of PBS and 25 ml of a 24-h BHIAmp culture ( C) containing 5 ml of each of the ve strains of E. coli O157:H7. Lettuce was submerged in the cell suspension for 1 min. Uninoculated lettuce submerged in PBS for 1 min served as a control. Lettuce was placed on a wire screen lined with hardware cloth and dried at C under a laminar ow hood for 1 h. Procedure for packaging. Cut lettuce (25 g) was placed in PD-961EZ lm bags (oxygen transmission rate: 7,000 cc/m 2 /24 h, carbon dioxide transmission rate: 21,000 cc/m 2 /24 h) (Cryovac Inc., Duncan, S.C.). Bags were heat sealed under ambient atmosphere. Lettuce was incubated for 0, 2, 4, 7, 10, 14, and 18 days at 58C and 0, 2, 4, and 7 days at 158C before analyzing for population of E. coli O157:H7. Effect of leaf thickness on E. coli O157:H7 attachment. Experiments were done to determine if the number of E. coli O157:H7 cells adhering to lettuce is different, depending on the thickness of the lettuce leaf. Lettuce leaves were divided into three groups ( mm, mm, and mm thick). Lettuce (25 g) from each group was dipped into PBS (4 liters) containing E. coli O157:H7 (10 7 CFU/ml) for 1 min, then dried under a laminar hood for 1 h. The number of CFU of E. coli O157:H7/g of lettuce was determined. Enumeration of E. coli O157:H7. Uninoculated lettuce and inoculated lettuce (25 g) subjected to various treatments and storage conditions were analyzed for populations of E. coli O157:H7. Each sample was combined with 225 ml of sterile Dey-Engley broth (Difco) in a sterile polyethylene bag and pummeled at medium speed with a stomacher (Seward Stomacher 400, London, UK) for 2 min. Duplicate samples of stomachate (0.1 ml) appropriately diluted in 0.1% peptone water were surface spread on BHIAmp and incubated at 378C for 24 h. Presumptive E. coli O157:H7 colonies were counted under UV light. Con rmation of E. coli O157:H7 was done using a latex agglutination kit (Oxoid, Hampshire, UK). Populations of E. coli O157:H7 in suspensions immediately before and after dipping lettuce were also determined. Statistical analysis. The Statistical Analysis System (27) using a general linear model and Pearson correlation was used to analyze data. The level of signi cance for all tests was a Mean separations were achieved using Duncan s multiple range test. All experiments were replicated four times. RESULTS AND DISCUSSION Effect of leaf thickness on attachment. The number of E. coli O157:H7 detected on inoculated lettuce was not signi cantly (a ) in uenced by leaf thickness. Leaves ranging from to mm thick contained a mean population of log 10 CFU/g. Inoculation before heat treatment. No E. coli O157: H7 was detected in the uninoculated lettuce. Populations of E. coli O157:H7 on lettuce inoculated before treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 58C are shown in Figure 1. Treatment with 20 ppm chlorine at either temperature did not result in significantly greater reductions in populations compared to respective treatments in water without chlorine. The pathogen steadily decreased throughout the storage of lettuce at 58C. While treatment reduced the number of CFU/g of lettuce compared to untreated (control) lettuce by 0.7 to 1.1 log 10 CFU/g, treatment did not affect patterns of inactivation during storage. Except for day 7, the population of E. coli

3 J. Food Prot., Vol. 64, No. 3 E. COLI O157:H7 IN LETTUCE 307 FIGURE 1. Populations of E. coli O157:H7 on lettuce inoculated before (not treated) and immediately after treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 58C for up to 18 days. O157:H7 detected in the untreated lettuce was signi cantly greater than number in treated lettuce stored for the same length of time. Populations of E. coli O157:H7 on lettuce inoculated before treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 158C for up to 7 days are shown in Figure 2. The population increased by 2.3 to 3.2 log 10 CFU/g within 2 days, then continued to increase at a slower rate through 7 days of storage. At 4 and 7 days, signi cantly higher populations were reached on lettuce treated at 508C, compared to samples that had been treated at 208C, regardless of whether treatment water contained 20 ppm chlorine. These results are in general agreement with previous studies. Hao and Brackett (17) studied the in uence of temperature and modi ed atmosphere on growth of E. coli O157:H7. They observed that E. coli O157:H7 grew at 10 and 208C and survived at 58C in tryptic soy broth. Abdul- Raouf et al. (1) reported that the population of E. coli O157: H7 on shredded lettuce stored at 58C signi cantly decreased during a 14-day storage period, whereas the pathogen grew on lettuce stored at 12 or 218C. In our study, a larger amount of cellular uid may have leaked from lettuce treated at 508C compared to 208C, thereby enhancing growth. This uid contains substantial amounts of simple sugars and other nutrients that can be utilized by E. coli O157:H7. Also, treatment at 508C may have reduced the number of microorganisms naturally present on lettuce, providing a less competitive environment for E. coli O157:H7 to grow. Chlorine is used as a disinfectant in wash, spray, and ume waters in the fresh fruit and vegetable industry. However, treatment of produce with chlorinated water for the purpose of drastically reducing populations of pathogens on FIGURE 2. Populations of E. coli O157:H7 on lettuce inoculated before (not treated) and immediately after treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 158C for up to 7 days. produce has not always been successful (10). Beuchat (6) reported that spraying lettuce with 200 ppm chlorine was not more effective in eliminating viable E. coli O157:H7 cells than spraying with water. Dipping Brussels sprouts containing about 6 log 10 CFU of Listeria monocytogenes into a 200-ppm chlorine solution for 10 s reduced the population of by about 2 log 10 CFU (9). Other researchers have reported that chlorine was minimally effective in reducing population of other pathogens on produce, e.g., L. monocytogenes in shredded lettuce (7) and cabbage (30), Salmonella Stanley on alfalfa seeds (18), and Salmonella Montevideo on mature green tomatoes (31). Populations of E. coli O157:H7 in the inoculum, treatment solution, and rinse solution, and on inoculated, treated lettuce are listed in Table 1. The number of E. coli O157: H7 in the inoculum did not change after it had been used to inoculate lettuce. The pathogen was detected in water without chlorine after dipping lettuce, which indicates that some of the E. coli O157:H7 cells were removed from lettuce during the dip treatment. E. coli O157:H7 was not detected in chlorinated water in which lettuce was dipped. Regardless of treatment temperature, the presence of 20 ppm chlorine in treatment solution did not kill all of the E. coli O157:H7 on lettuce as evidenced by detection of 3.3 and 3.4 log 10 CFU/g of lettuce after treatment at 50 or 208C, respectively, and rinsing at 108C. The lethal activity of chlorine depends on the amount of free available chlorine (hypochlorous acid, HOCl) that comes in contact with microbial cells (5). The active form of HOCl is rapidly neutralized upon contact with organic material. Thus, the ef cacy of chlorine in killing microorganisms on the surface of raw produce is markedly less than in systems such as the dipping solution, which is free of organic matter. E. coli O157:H7 can penetrate into the

4 308 LI ET AL. J. Food Prot., Vol. 64, No. 3 TABLE 1. Population of E. coli O157:H7 in inoculum, water, treatment solution, and rinse solution, and on lettuce after inoculation, treatment and rinse Inoculum, treatment solution, or rinse solution Log 10 CFU/ml of inoculum, water treatment, solution, or rinse solution Log 10 CFU/g of lettuce Inoculum Before inoculation After inoculation Treatment (508C) Without chlorine Water With 20 ppm chlorine Treatment solution Treatment (208C) Without chlorine Water With 20 ppm chlorine Treatment solution a Less than 1 CFU/ml a stomata and cut edges of lettuce leaves (26), resulting in protection against contact with chlorine (28). Hydrophobic pockets or crevices in the leaf surface may also provide protection (3, 8). This limits the antimicrobial effectiveness of chlorinated water treatment of lettuce as well as other raw produce. In addition to the type of produce and target microorganism, other factors affecting the ef cacy of chlorine as a disinfectant include time of exposure, temperature, and ph (9, 30). In our study, we treated lettuce in water containing 20 ppm chlorine and for 90 s only. Higher treatment temperature or extended duration of exposure to heat may have killed larger numbers of E. coli O157:H7. However, Loaiza-Valarde et al. (20) observed that treatment of lettuce at 558C for 60 s caused deterioration of lettuce tissue, thus perhaps limiting the practicality of treatment at a temperature between 50 and 558C. Inoculation after heat treatment. Populations of E. coli O157:H7 on lettuce inoculated after treatment for 90 s at 50 or 208C in water with or without 20 ppm chlorine, and chilling at 108C, followed by storing at 58C are shown in Figure 3. As with lettuce inoculated before treatment, then chilled at 108C, and stored at 58C (Fig. 1), the number of E. coli O157:H7 decreased signi cantly (1.3 to 1.9 log 10 CFU/g) during storage for up to 18 days. The increases in counts after 2 days on lettuce treated at 50 or 208C in water without chlorine were signi cant. Populations on lettuce stored for the same number of days were not signi cantly different, regardless of treatment temperature. Reasons for FIGURE 3. Populations of E. coli O157:H7 on lettuce inoculated after treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 58C for up to 18 days. the increase in counts during the rst 2 days of storage on lettuce not treated with chlorine are not evident. Shown in Figure 4 are populations of E. coli O157:H7 on lettuce inoculated after treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 158C for up to 7 days. Populations increased signi cantly (2.2 to 2.6 log 10 CFU/g) within 2 days, regardless of treatment. As observed in the rst series of experiments in which lettuce was inoculated before treatment (Fig. 2), a trend of higher populations occurred on lettuce treated at 508C compared to 208C, regardless of chlorine treatment. This again indicates that treatment of lettuce at 508C may enhance subsequent growth of E. coli O157:H7. FIGURE 4. Populations of E. coli O157:H7 on lettuce inoculated after treatment at 50 or 208C in water with or without 20 ppm chlorine, then stored at 158C for up to 7 days.

5 J. Food Prot., Vol. 64, No. 3 E. COLI O157:H7 IN LETTUCE 309 Heat treatment has promise in reducing the activity of enzymes that cause browning of lettuce, thereby extending the shelf life (20). However, our study indicates that heat treatment does not inhibit, but rather enhances, the growth of E. coli O157:H7 on cut lettuce stored at 158C. Treatment of lettuce with 20 ppm chlorine at 50 or 208C before or after inoculation with E. coli O157:H7 did not have a marked in uence on behavior of the pathogen during subsequent storage at 5 or 158C. ACKNOWLEDGMENT The authors sincerely acknowledge Dr. Ying Mao for her technical help. REFERENCES 1. Abdul-Raouf, U. M., L. R. Beuchat, and M. S. Ammer Survival and growth of Escherichia coli O157:H7 on salad vegetables. Appl. Environ. Microbiol. 7: Ackers, M., B. Majon, E. Leahy, T. Damrow, L. Hutwagner, T. Barrett, W. Bibb, P. Hayes, P. Grif n, and L. Slutsker An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce consumption, Western Montana, p. 258, K43. Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother. American Society of Microbiology, Washington, D.C. 3. Adams, M. R., A. D. Harley, and L. J. Cox Factors affecting the ef cacy of washing procedures used in the production of prepared salads. Food Microbiol. 6: Beuchat, L. R Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59: Beuchat, L. R Surface decontamination of fruits and vegetables eaten raw: a review. Food Safety Unit, World Health Organization. WHO/FSF/FOS/98.2, 42 p. 6. Beuchat, L. R Survival of Escherichia coli O157:H7 in bovine feces applied to lettuce and the effectiveness of chlorine water as a disinfectant. J. Food Prot. 62: Beuchat, L. R., and R. E. Brackett Survival and growth of Listeria monocytogenes on lettuce as in uenced by shredding, chlorine treatment, modi ed atmosphere packaging and temperature. J. Food Sci. 55: , Beuchat, L. R., B. V. Nail, B. B. Adler, and M. R. S. Clavero Ef cacy of spray application of chlorinated water in killing pathogenic bacterial on raw apples, tomatoes, and lettuce. J. Food Prot. 61: Brackett, R. E Antimicrobial effect of chlorine on Listeria monocytogenes. J. Food Prot. 50: Brackett, R. E Incidence, contributing factors, and control of bacterial pathogens in produce. Postharvest Biol. Technol. 15: Brecht, J. K Physiology of lightly processed fruits and vegetables. Hortic. Sci. 301: Delaquis, P. J., S. Stewart, P. M. A. Toivonen, and A. L. Moyls Effect of warm, chlorinated water on the microbial ora of shredded iceberg lettuce. Food Res. Int. 32: De Roever, C Microbiological safety evaluations and recommendations on fresh produce. Food Control 9: Doyle, M. P., T. Zhao, J. Meng, and P. Zhao Escherichia coli O157:H7, p In M. P. Doyle, L. R. Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals and frontiers. American Society of Microbiology Press, Washington, D.C. 15. Eckert, J. W., and J. M. Ogawa The chemical control of postharvest diseases: deciduous fruits, berries, vegetables and root/tuber crops. Annu. Rev. Phytopathol. 26: Francis, G. A., C. Thomas, and D. O Beirne The microbiological safety of minimally processed vegetables. Int. J. Food Sci. Technol. 34: Hao, Y.-Y., and R. E. Brackett Growth of Escherichia coli O157:H7 in modi ed atmosphere. J. Food Prot. 56: Jaquette, C. B., L. R. Beuchat, and B. E. Mahon Ef cacy of chlorine and heat treatment in killing Salmonella Stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage. Appl. Environ. Microbiol. 62: Ke, D., and M. E. Saltveit Wound induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce. Physiol. Plant. 76: Loaiza-Valarde, J. G., F. A. Tomás-Barberá, and M. E. Saltveit Effects of intensity and duration of heat-shock treatments on woundinduced phenolic metabolism in iceberg lettuce. J. Am. Soc. Hortic. Sci. 122: López-Gálvez, G., M. E. Saltveit, and M. Cantwell Woundinduced phenylalanine minimally processed lettuce. Postharvest Biol. Technol. 9: Mermim, J., P. Mead, K. Gensheimer, and P. Grif n Outbreak of Escherichia coli O157:H7 infections among boy scouts in Maine, p. 258, K44. Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother. American Society of Microbiology, Washington, D.C. 23. Nguyen-the, C., and F. Carlin Fresh and processed vegetables, p In B. M. Lund, A. C. Baird-Parker, and G. W. Gould (ed.), The microbiological safety and quality of food, vol. 1. Aspen Publ., Gaithersburg, Md. 24. Rice, E. W., R. M. Clark, and C. H. Johnson Chlorine inactivation of Escherichia coli O157:H7. Emerg. Infect. Dis. 5: Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargett, P. A. Blake, and M. L. Cohen Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308: Seo, K. H., and J. F. Frank Attachment of Escherichia coli O157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy. J. Food Prot. 62: Stastistical Analysis Sysytems SAS user s guide: statistics. SAS Institute Inc., Cary, N.C. 28. Takeuchi, K., and J. F. Frank Penetration of Escherichia coli O157:H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. J. Food Prot. 63: Taormina, P. T., and L. R. Beuchat Comparison of chemical treatments to eliminate enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds. J. Food Prot. 62: Zhang, S., and J. M. Farber The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiol. 13: Zhuang, R.-Y., L. R. Beuchat, and F. J. Angulo Fate of Salmonella Montevideo on and in raw tomatoes as affected by temperature as treatment with chlorine. J. Food Prot. 61: