Mild heat treatment of lettuce enhances growth of Listeria monocytogenes during subsequent storage at 5 C or 15 C

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1 Journal of Applied Microbiology 2002, 92, Mild heat treatment of lettuce enhances growth of Listeria monocytogenes during subsequent storage at 5 C or 15 C Y. Li 1, R.E. Brackett 1,2, J. Chen 1 and L.R. Beuchat 1 1 Center for Food Safety and Department of Food Science and Technology, University of Georgia and 2 Office of Plant and Dairy Foods and Beverages, US Food and Drug Administration, Washington, D.C., USA 2001/155: received 26 June 2001, revised 9 August 2001 and accepted 15 August 2001 Y. L I, R. E. B R A C K E T T, J. C H E N A N D L.R. B E U C H A T Aims: The objective of this study was to determine the influence of mild heat treatment, storage temperature and storage time on the survival and growth of Listeria monocytogenes inoculated onto cut iceberg lettuce leaves. Methods and Results: Before or after inoculation with L. monocytogenes, cut iceberg lettuce leaves were dipped in water (20 or 50 C), containing or not 20 mg l 1 chlorine, for 90 s, then stored at 5 C for up to 18 days or 15 C for up to 7 days. The presence of 20 mg l 1 chlorine in the treatment water did not significantly (a ¼ 0Æ05) affect populations of the pathogen, regardless of other test parameters. The population of L. monocytogenes on lettuce treated at 50 C steadily increased throughout storage at 5 C for up to 18 days. At day 10 and thereafter, populations were 1Æ7 2Æ3 log 10 cfu g 1 higher on lettuce treated at 50 C after inoculation compared with untreated lettuce or lettuce treated at 20 C, regardless of chlorine treatment. The population of L. monocytogenes increased rapidly on lettuce stored at 15 C. At 2 and 4 days, significantly higher populations were detected on lettuce that had been treated at 50 C, compared with respective samples that had been treated at 20 C, regardless of inoculation before or after treatment, or the presence of 20 mg l 1 chlorine in the treatment water. Conclusions: The results clearly demonstrated that mild heat treatment of cut lettuce leaves enhances the growth of L. monocytogenes during subsequent storage at 5 or 15 C. Significance and Impact of the Study: Mild heat treatment of cut lettuce may result in a prolonged shelf life as a result of delaying the development of brown discoloration. However, heat treatment also facilitates the growth of L. monocytogenes during storage at refrigeration temperature, thereby increasing the potential risk of causing listeriosis. INTRODUCTION Listeria monocytogenes has been isolated from a wide range of raw vegetables (Berrang et al. 1989; Beuchat and Brackett 1991; Carlin and Nguyen-the 1994; Carlin et al. 1995; Beuchat 1996a, b; Aureli et al. 2000). A major outbreak of listeriosis was associated with the consumption of coleslaw in Canada in 1981 (Schlech et al. 1983), and lettuce, celery and tomatoes were implicated in an outbreak of listeriosis in eight Boston hospitals (Ho et al. 1986). Because of the Correspondence to: L.R. Beuchat, Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, Georgia , USA ( lbeuchat@cfs.griffin.peachnet.edu). widespread distribution of L. monocytogenes in soil and on plant surfaces, it is important to understand the ability of the pathogen to survive and grow under conditions associated with processing, distribution and storage of raw fruits and vegetables. Lettuce is a common vegetable in many countries. It is most often consumed raw, especially in salads and sandwiches. Production and consumption of minimally-processed (ready-to-eat) lettuce have increased dramatically in many countries in recent years. The convenience of cut, prewashed, packaged lettuce benefits consumers and has created a demand for high quality products. However, cutting lettuce leaves can induce biochemical reactions that cause deterioration of sensory quality (Brecht 1995). Wounded tissue responses include increased phenylalanine ammonia ª 2002 The Society for Applied Microbiology

2 270 Y. LI ET AL. lyase (PAL) activity, which initiates the biosynthesis of phenylpropanoid products (Ke and Saltveit 1989). These phenolic compounds can be oxidized by polyphenol oxidase (PPO) to form brown-coloured compounds which lead to visible discoloration along the cut edge of lettuce leaves (López-Gálvez et al. 1996). Researchers have observed that dipping fresh-cut lettuce in water at 45 or 55 C inhibits activity of PAL (Loaiza- Valarde et al. 1997). Delaquis et al. (1999) described the effect of warm, chlorinated water on naturally-existing microbiota on shredded iceberg lettuce. Li et al. (2001a) studied the survival and growth of Escherichia coli O157 : H7 inoculated onto cut lettuce before or after heating in chlorinated water. Mild heat treatment retarded discoloration but growth of E. coli O157 : H7 was enhanced. Investigations to determine the effect of mild heat treatment on survival and growth of L. monocytogenes have not been reported. The study reported here was designed to determine the effects of heat treatment, storage temperature and storage time on the survival and growth of L. monocytogenes inoculated onto cut iceberg lettuce. The efficacy of treatment of lettuce with 20 mg l 1 free chlorine in killing the pathogen was also determined. MATERIALS AND METHODS Strains of L. monocytogenes and preparation of inoculum Five strains of Listeria monocytogenes, F8027 (serotype 4b, from celery), H0222 (serotype 1/2a, from potato), F8255 (serotype 1/2b, from peach/plum), G1091 (serotype 4b, from a coleslaw outbreak) and F8369 (serotype 1/2a, from corn) were grown in 10 ml Brain Heart Infusion (BHI; BD Diagnostic Systems, Milwaukee, WI, USA) broth for 18 h at 37 C. Three successive 18 h transfers to 10 ml BHI broth were made prior to preparing inocula. A mixture with equal numbers of cells of each strain was used as an inoculum. Preparation for chlorinated water Distilled water containing 20 mg l 1 free chlorine was prepared by combining water with sodium hypochlorite (Aldrich Chemical Co., Inc., Sparks, MD, USA). The ph was adjusted to 7Æ0 ± 0Æ02 using hydrochloric acid. A Hach test kit (model CN-66, Hach Company, Loveland, CO, USA) was used to quantify free chlorine. Preparation of cut lettuce Iceberg lettuce (Lactuca sativa L.) was purchased from a local grocery store in Griffin, GA. Lettuce was stored at 5 C after purchase and used in experiments within 24 h. Two or three outer leaves and the core were removed and discarded. Lettuce leaves were cut into pieces using a sterilized circular cookie cutter (6Æ5 cm diameter). Very thin or thick pieces were not used in experiments. Procedures for inoculation and treatment To mimic contamination at two points during processing, lettuce was inoculated before and after treatment at 50 or 20 C with 20 mg l 1 chlorine. The first series of experiments involved inoculation before treatment. This would simulate contamination of lettuce before washing with chlorinated water in the field or at a processing plant. Twelve litres of 0Æ1 mol l 1 potassium phosphate buffer solution (PB, ph 7Æ0) were combined with 50 ml of a mixture BHI broth cultures of the five strains of L. monocytogenes. Pieces of cut lettuce leaves (1500 g) were submerged with gentle agitation in the cell suspension (22 ± 2 C) for 1 min. Lettuce submerged in PB for 1 min served as a control (uninoculated). Lettuce was placed on a wire screen lined with a coarsely woven cloth to dry at 22 ± 2 C under a laminar flow hood for 1 h. Inoculated or uninoculated lettuce (270 g) was then placed in a stainless steel screen basket and submerged for 90 s in a water bath containing 4 l of warm (50 C) distilled water with chlorine (20 mg l 1 ) or without chlorine, or 4 l of water at 20 C with chlorine (20 mg l 1 ) or without chlorine, then drained. Lettuce was submerged in 4 l of water at 10 C for 30 s, immediately after treatment at 50 or 20 C in chlorinated or non-chlorinated water, before drying under a laminar flow hood at 22 ± 2 C for 30 min. A second series of experiments involved inoculation of lettuce with L. monocytogenes after treatment with chlorine. This procedure simulated contamination of cut lettuce at some point after washing with chlorinated water in the field or a processing plant. Cut lettuce (270 g) was placed in a stainless steel screen basket and submerged in 4 l distilled water (20 or 50 C) containing 20 mg l 1 chlorine or no chlorine for 90 s, then drained as described in the first series of experiments. Lettuce was then submerged in 4 l distilled water at 10 C for 30 s before combining with 6 l PB and 25 ml of a mixture of BHI broth cultures of the five strains of L. monocytogenes. Lettuce was submerged in the cell suspension for 1 min. Lettuce submerged for 1 min in PB not containing L. monocytogenes served as a control. Inoculated and uninoculated lettuce was placed on a wire screen lined with coarsely woven cloth and dried at 22 ± 2 C under a laminar flow hood for 1 h. Procedure for packaging Inoculated and uninoculated lettuce (25 g) was placed in PD-961EZ film bags (oxygen transmission rate:

3 LISTERIA MONOCYTOGENES IN LETTUCE cc m 2 24 h 1, carbon dioxide transmission rate: cc m 2 24 h 1 ) (Cryovac Inc., Duncan, SC, USA). Bags were heat-sealed under ambient atmosphere. Lettuce was incubated for 0, 2, 4, 7, 10, 14 and 18 days at 5 C, or 0, 2, 4 and 7 days at 15 C, before analysing for populations of L. monocytogenes. Enumeration of L. monocytogenes Uninoculated lettuce, and inoculated lettuce subjected to various treatments and storage conditions, was analysed for populations of L. monocytogenes. Each sample (25 g) was combined with 225 ml sterile Dey-Engley (D/E) neutralizing broth (BD Diagnostic Systems) in a sterile polyethylene bag and pummeled at high speed with a stomacher 400 (Seward Medical Ltd, London, UK) for 2 min. Duplicate samples of stomachate (0Æ1 ml) appropriately diluted in sterile 0Æ1% peptone water were surface spread on MOX agar (Oxford Medium Base with Modified Oxford Antimicrobic Supplement; BD Diagnostic Systems) and incubated at 37 C for 48 h. Presumptive L. monocytogenes colonies were counted. Colonies were randomly picked and subjected to biochemical confirmation tests, including Gram-stain reaction, motility on motility test medium, haemolysis on horse blood agar and API-Listeria (bio- Mérieux, Hazelwood, MO, USA) assay strips. Uninoculated lettuce samples were analysed for the presence of L. monocytogenes in Listeria Enrichment Broth (Oxoid) for 48 h at 30 C. Cultures were streaked on MOX agar, incubated for 48 h at 37 C and examined for presumptive L. monocytogenes colonies. Confirmation of selected colonies was carried out as described above. PCR analysis To determine whether one or more of the five strains was dominant in lettuce after storage for 18 days at 5 C or 7 days at 15 C, three colonies from selected plates containing colonies were subjected to PCR analysis. Cells were grown on BHI agar at 37 C for h. Colonies were transferred with a sterile loop into 200 ll BHI broth and cultured at 37 C for h with vigorous shaking (150 rev min 1 ) on a rotary shaker. Crude DNA was prepared by boiling bacterial cell suspensions; 200 ll of culture was centrifuged (model 5415C Microcentrifuge, Eppendorf, Hamburg, Germany) for 2 min at g. Pellets were washed in 200 ll sterile deionized water and centrifuged as described above. The cell suspension was boiled for 10 min; 20 ll supernatant fluid were used as a template for PCR. A 22 base oligonucleotide primer ERIC1R (5 -ATG TAA GCT CCT GGG GAT TCAC-3 ) designed by Versalovic et al. (1991) was used. PCR assays were performed with a DNA thermal cycler (model 480, Perkin-Elmer, Norwalk, CT, USA) using one cycle at 94 C for 5 min, followed by 40 cycles at 92 C for 45 s, 25 C for 1 min, 68 C for 10 min, and final extension at 72 C for 20 min. The PCR amplicons were analysed by gel electrophoresis on a 1% agarose (Gibco BRL, Rockville, MD, USA) gel in TBE buffer (0Æ089 mol l 1 Tris-borate, 0Æ002 mol l 1 EDTA, ph 8Æ0). The gel was stained with ethidium bromide and visualized with the Gel Doc System 2000 (Bio-Rad laboratories, Hercules, CA, USA). Statistical analysis All experiments were replicated four times. The Statistical Analysis System (SAS 1987) using a general linear model (GLM) and Pearson correlation was used to analyse data. The level of significance reported for all tests is a ¼ 0Æ05. Mean separations were achieved using Duncan s Multiple Range test. RESULTS Inoculation before heat treatment Listeria monocytogenes was not detected in the uninoculated lettuce. Populations of L. monocytogenes on inoculated, untreated lettuce and lettuce inoculated before treatment at 50 or 20 C in water, with or without 20 mg l 1 chlorine, then stored at 5 C, are shown in Fig. 1. All treatments reduced initial populations of L. monocytogenes by 1Æ0 1Æ2 log 10 cfu g 1. However, treatment of inoculated lettuce with 20 mg l 1 chlorine at 50 or 20 C did not result in significant (a ¼ 0Æ05) reductions in populations compared with respective treatments in water without chlorine. At day 10 and thereafter, populations of L. monocytogenes on lettuce treated at 50 C were 1Æ7 2Æ3 log 10 cfu g 1 higher than populations on untreated lettuce or lettuce treated at 20 C, regardless of the presence of 20 mg l 1 chlorine in the treatment water. Populations of L. monocytogenes on lettuce inoculated before treatment at 50 or 20 C in water with or without 20 mg l 1 chlorine, then stored at 15 C for up to 7 days, are shown in Fig. 2. The population increased rapidly during the first 2 days of storage, then remained relatively unchanged through 7 days. Increases were more rapid on lettuce treated at 50 C compared with increases on lettuce treated at 20 C. At 2 and 4 days, significantly higher populations of L. monocytogenes were detected on lettuce treated at 50 C, compared with lettuce that had been treated at 20 C, regardless of treatment with water containing 20 mg l 1 chlorine. This trend continued through 7 days of storage. Populations at days were about 5Æ0and6Æ0log 10 cfu g 1 on lettuce not treated or treated with 20 mg l 1, respectively. The shelf life of commercial cut ready-to-eat lettuce properly

4 272 Y. LI ET AL Fig. 1 Populations of Listeria monocytogenes on lettuce inoculated before treatment at 50 or 20 C in water with or without 20 mg l 1 chlorine, then stored at 5 C for up to 18 days. (r), Not treated; (d), 50 C with chlorine; (m), 20 C with chlorine; (s), 50 C without chlorine; (n), 20 C without chlorine Inoculation after heat treatment Listeria monocytogenes was not detected in the uninoculated lettuce. Populations of L. monocytogenes on lettuce inoculated after treatment for 90 s at 50 or 20 C in water, with or without 20 mg l 1 chlorine, followed by storing at 5 C, are shown in Fig. 3. Populations on inoculated untreated lettuce are also shown. In treated lettuce, the number of L. monocytogenes increased by 0Æ6 1Æ2 log 10 cfu g 1 during storage for up to 18 days. Populations on lettuce receiving various treatments and stored for the same length of time were not significantly different. Populations on untreated lettuce were essentially unchanged during the 18 day storage period. Shown in Fig. 4 are populations of L. monocytogenes on untreated lettuce and on lettuce inoculated after treatment at 50 or 20 C in water, with or without 20 mg l 1 chlorine, then stored at 15 C for up to 7 days. Populations increased 1Æ5 2Æ3 log 10 cfu g 1 within 2 days on untreated and treated lettuce. At day 2, significantly higher populations occurred on lettuce treated at 50 C compared with lettuce treated at 20 C, regardless of chlorine treatment. Treatment of lettuce at 50 C before inoculation with L. monocytogenes and storage at 15 C clearly enhanced growth. Higher populations were achieved on lettuce treated in chlorinated water at 50 C compared with populations on lettuce treated at 20 C Fig. 2 Populations of Listeria monocytogenes on lettuce inoculated before treatment at 50 or 20 C in water with or without 20 mg l 1 chlorine, then stored at 15 C for up to 7 days. (r), Not treated; (d), 50 C with chlorine; (m), 20 C with chlorine; (s), 50 C without chlorine; (n), 20 C without chlorine stored at 5 C generally does not exceed 12 days and may be as short as 8 days, depending on initial populations of naturally-occurring micro-organisms. Fig. 3 Populations of Listeria monocytogenes on lettuce inoculated after treatment at 50 or 20 C in water with or without 20 mg l 1 chlorine, then stored at 5 C for up to 18 days. (r), Not treated; (d), 50 C with chlorine; (m), 20 C with chlorine; (s), 50 C without chlorine; (n), 20 C without chlorine

5 LISTERIA MONOCYTOGENES IN LETTUCE Fig. 4 Populations of Listeria monocytogenes on lettuce inoculated after treatment at 50 or 20 C in water with or without 20 mg l 1 chlorine, then stored at 15 C for up to 7 days. (r), Not treated; (d), 50 C with chlorine; (m), 20 C with chlorine; (s), 50 C without chlorine; (n), 20 C without chlorine PCR analysis Results of PCR analysis are shown in Table 1. A total of 108 isolates were examined. On lettuce inoculated before heat treatment then stored at 15 C for 7 days, stain F8255 was predominant (60% of isolates), while F8369 was predominant (30%) on lettuce stored for 18 days at 5 C. Strain F8027 was predominant (50% at 5 C and 75% at 15 C) on lettuce inoculated with L. monocytogenes after heat treatment. DISCUSSION Chlorinated water is not as effective in killing pathogens on raw fruits and vegetables as might be expected based on the lethal activity observed in simple aqueous systems. The use of chlorine and other chemicals in wash water has been shown to reduce populations of pathogens on raw vegetables. However, elimination cannot been assured (Beuchat 1998; Francis et al. 1999). Reduction in populations of L. monocytogenes on fresh-cut lettuce treated with 200 mg l 1 chlorine was reported to be 1Æ3 1Æ7 log 10 cfu g 1 (Zhang and Farber 1996). In vitro experiments (Brackett 1987; El-Kest and Marth 1988) have shown that the action of chlorine against L. monocytogenes occurs primarily during the first 30 s of exposure. In the present study, populations of L. monocytogenes on inoculated lettuce decreased by about 1 log 10 cfu g 1 after treatment in water, regardless of temperature or the presence of chlorine. Growth of L. monocytogenes on lettuce heated at 50 C was enhanced, compared with growth on lettuce treated at 20 C, regardless of subsequent storage at 5 or 15 C. Enhanced growth may result from reduction in numbers of competitive background micro-organisms that might otherwise compete with L. monocytogenes. Li et al. (2001b) observed that populations of mesophilic aerobic microorganisms, psychrotrophs and Enterobacteriaceae were reduced on lettuce subjected to the same treatments as those used in the present study. Tissue fluid and residual water from treatments may also enhance the rate of growth of L. monocytogenes. The pathogen has been reported to grow at 5 C on inoculated lettuce stored for 14 days (Steinbruegge et al. 1988). Beuchat and Brackett (1990), in contrast, reported that populations of L. monocytogenes on unwashed, shredded lettuce were essentially unchanged throughout 15 days of storage at 5 C. Other researchers have reported that L. monocytogenes grows at refrigeration temperature on fresh-cut vegetables (Garcia-Gimeno and Zurera-Cosano 1997; Farber et al. 1998), endive (Carlin et al. 1996) and shredded cabbage (Kallander et al. 1991). The strain isolated from celery (strain F8027) grew to higher numbers than the other four strains on refrigerated lettuce. Compared with other strains, higher numbers of strain F8255, from a peach/plum, were recovered from Table 1 Frequency of stains of Listeria monocytogenes isolated from lettuce stored at 5 or 15 C Storage Strain number Inoculation* Temperature ( C) Time (days) F8027 H0222 F8255 G1091 F8369 Total Before After Total *Lettuce was inoculated with L. monocytogenes before or after all treatments. Number of colonies confirmed by PCR.

6 274 Y. LI ET AL. lettuce heated at 50 C. The coleslaw outbreak strain (G1091) appeared to be the least able of the five test strains to survive and grow on cut lettuce. Further studies need to be conducted to determine the reasons for, and practical implications of differences in the ability of test strains to survive and grow in cut lettuce. Raw vegetables are occasionally contaminated with L. monocytogenes (Farber and Peterkin 1991; Nguyen-the and Carlin 1994; Beuchat 1996a). Transmission of L. monocytogenes to vegetables can occur via manure, sewage sludge, irrigation water, or by direct contamination from faeces of birds and animals. Contamination may also result from lack of hygienic handling or storage practices (Beuchat 1996b; Beuchat and Ryu 1997). Francis and O Beirne (1997) observed that process operations have the potential to increase survival and growth of L. monocytogenes on vegetables, particularly when vegetables are subjected to mild temperature abuse. In the present study, mild heat treatment may have affected L. monocytogenes indirectly by releasing tissue fluids, thus increasing nutrient availability for growth. These and other observations emphasize the importance of strict hygiene during production, processing and packaging to avoid contamination of lettuce before and after washing or sanitizing. In conclusion, although mild heat treatment of cut lettuce may result in a prolonged shelf life as a result of delaying browning reactions (Loaiza-Valarde et al. 1997; Li et al. 2001a), it also facilitates conditions that promote growth of L. monocytogenes, thereby increasing the potential risk of causing listeriosis. REFERENCES Aureli, P., Fiorucci, G.C., Caroli, D. et al. (2000) An outbreak of febrile gastroenteritis associated with corn contaminated by Listeria monocytogenes. New England Journal of Medicine 342, Berrang, M.E., Brackett, R.E. and Beuchat, L.R. (1989) Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere. Journal of Food Protection 52, Beuchat, L.R. (1996a) Listeria monocytogenes: Incidence on vegetables. Food Control 7, Beuchat, L.R. (1996b) Pathogenic microorganisms associated with fresh produce. Journal of Food Protection 59, Beuchat, L.R. (1998) Surface decontamination of fruits and vegetables eaten raw: a review. Bulletin WHO/FSF/FOS/98.2. Beuchat, L.R. and Brackett, R.E. (1990) Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. Journal of Food Science 55, , 870. Beuchat, L.R. and Brackett, R.E. (1991) Behavior of Listeria monocytogenes inoculated into raw tomatoes and processed tomato products. Applied and Environmental Microbiology 57, Beuchat, L.R. and Ryu, J.-H. (1997) Produce handling and processing practice. Emerging Infectious Diseases 3, Brackett, R.E. (1987) Antimicrobial effect of chlorine on Listeria monocytogenes. Journal of Food Protection 50, Brecht, J.K. (1995) Physiology of lightly processed fruits and vegetables. Hortscience 301, Carlin, F. and Nguyen-the, C. (1994) Fate of Listeria monocytogenes on four types of minimally processed green salads. Letters in Applied Microbiology 18, Carlin, F., Nguyen-the, C. and Abreu da Silva, A. (1995) Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive. Journal of Applied Bacteriology 78, Carlin, F., Nguyen-the, C., Abreu da Silva, A. and Cochet, C. (1996) Effects of carbon dioxide on the fate of Listeria monocytogenes of aerobic bacteria and on the development of spoilage in minimally processed fresh endive. International Journal of Food Microbiology 32, Delaquis, P.T., Stewart, S., Toivonon, P.M.A. and Moyls, A.L. (1999) Effect of warm, chlorinated water on microbial flora of shredded iceberg lettuce. Food Research International 32, El-Kest, S.E. and Marth, E.H. (1988) Inactivation of Listeria monocytogenes by chlorine. Journal of Food Protection 51, Farber, J.M. and Peterkin, P.I. (1991) Listeria monocytogenes, a foodborne pathogen. Microbiology Reviews 55, Farber, J.M., Wang, S.L., Cai, Y. and Zhang, S. (1998) Changes in populations of Listeria monocytogenes inoculated on packaged fresh cut vegetables. Journal of Food Protection 61, Francis, G.A. and O Beirne, D. (1997) Effects of gas atmosphere, antimicrobial dip and temperature on the fate of Listeria innocua and Listeria monocytogenes on minimally processed lettuce. International Journal of Food Science and Technology 32, Francis, G.A., Thomas, C. and O Beirne, D. (1999) The microbiological safety of minimally processed vegetable. International Journal of Food Science and Technology 34, Garcia-Gimeno, R. and Zurera-Cosano, G. (1997) Determination of ready-to-eat vegetable salad shelf-life. International Journal of Food Microbiology 36, Ho, J.L., Shades, K.N., Friedland, G., Eckind, P. and Fraser, D.W. (1986) An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Archives of International Medicine 146, Kallander, K.D., Hitchins, A.D., Lancette, G.A. et al. (1991) Fate of Listeria monocytogenes in shredded cabbage stored at 5 and 25 C under a modified atmosphere. Journal of Food Protection 54, Ke, D. and Saltveit, M.E. (1989) Wound induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce. Physiologia Planterum 76, Li, Y., Brackett, R.E., Chen, J. and Beuchat, L.R. (2001a) 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. Journal of Food Protection 63, Li, Y., Brackett, R.E., Shewfelt, R.L. and Beuchat, L.R. (2001b) Changes in appearance and natural microflora on iceberg lettuce treated in warm, chlorinated water and then stored at refrigeration temperature. Food Microbiology 18, Loaiza-Valarde, J.G., Tomás-Barberá, F.A. and Saltveit, M.E. (1997) Effects of intensity and duration of heat-shock treatments on woundinduced phenolic metabolism in iceberg lettuce. Journal of the American Society for Horticultural Science 122,

7 LISTERIA MONOCYTOGENES IN LETTUCE 275 López-Gálvez, G., Saltveit, M.E. and Cantwell, M. (1996) Woundinduced phenylalanine ammonia lyase activity: factors affecting its induction and correlation with quality of minimally processed lettuce. Postharvest Biology and Technology 9, Nguyen-the, C. and Carlin, F. (1994) The microbiology of minimally processed fresh fruits and vegetables. Critical Reviews in Food Science and Nutrition 34, SAS (1987) SAS User s Guide: Statistics. Cary, NC, USA: SAS Institute Inc. Schlech, W.F., Lavigne, P.M., Bortolussi, R.A. et al. (1983) Epidemic listeriosis evidence for transmission by food. New England Journal of Medicine 308, Steinbruegge, E.G., Maxcy, B.R. and Liewen, M.B. (1988) Fate of Listeria monocytogenes on ready to serve lettuce. Journal of Food Protection 51, Versalovic, J., Koeuth, T. and Lupski, J.R. (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 19, Zhang, S. and Farber, J.M. (1996) The effects of various disinfections against Listeria monocytogenes on fresh-cut vegetables. Food Microbiology 13,