Survival and growth of Listeria monocytogenes and Escherichia coli O157:H7 in ready-to-eat iceberg lettuce washed in warm chlorinated water

Cut iceberg lettuce inoculated with Escherichia coli O157:H7 and Listeria monocytogenes before and after washing for 3 min in cold (4 degrees C) and warm (47 degrees C) water containing 100 mg/liter total chlorine was stored at I and 10 degrees C in oxygen-permeable film packages (6,000 to 8,000 cc/m2/24 h). Cold chlorinated water was detrimental to the survival of E. coli O157: H7 and L. monocytogenes at both storage temperatures. In contrast, washing in warm chlorinated water favored the growth of both pathogens in lettuce stored at 10 degrees C. There was no evidence of a relationship between the magnitude of spoilage microflora and the fate of either bacterium.     

Survival of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella in juice concentrates

The survival of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella was studied in apple, orange, pineapple, and white grape juice concentrates and banana puree. Pouches of juice concentrate or puree were inoculated with pathogens at a level > or = 10(3) CFU/g and stored at -23 degrees C (-10 degrees F). Pathogen survival was monitored at 6 and 24 h, once a week for four consecutive weeks, and biweekly thereafter until 12 weeks. When pathogens were not detectable by direct plating, samples were enriched in universal preenrichment broth for 72 h and plated on selective media. Results showed that E. coli O157:H7, L. monocytogenes, and Salmonella were recoverable from all five concentrates through 12 weeks of storage at -23 degrees C.     

Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Pseudomonas fluorescens to lettuce leaves

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.     

Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR

A protocol enabling simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains was devised and evaluated using artificially contaminated fresh produce. Association of Official Analytical Chemists (AOAC)-approved polymerase chain reaction (PCR) detection methods for three human pathogens were modified to enable simultaneous and real-time detection with high throughput capability. The method includes a melting-curve analysis of PCR products, which serves as confirmatory test. The modified protocol successfully detected all three pathogens when fresh produce was washed with artificially contaminated water containing E. coli O157:H7 and S. typhimurium down to the predicted level of 1 to 10 cells/ml and L. monocytogenes at 1000 cells/ml. The ability to monitor several pathogens simultaneously will save time and increase our ability to assure food safety.     

Survival of Escherichia coli O157:H7, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes in Kimchi

The survival of gram-positive and gram-negative foodborne pathogens in both commercial and laboratory-prepared kimchi (a traditional fermented food widely consumed in Japan) was investigated. It was found that Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes could survive in both commercial and laboratory-prepared kimchi inoculated with these pathogens and incubated at 10 degrees C for 7 days. However, when incubation was prolonged, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, whereas Salmonella Enteritidis and L. monocytogenes took 16 days to reach similar levels in commercial kimchi. On the other hand, E. coli O157:H7 remained at high levels throughout the incubation period. For laboratory-prepared kimchi, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, and L. monocytogenes took 20 days to reach a similar level. E. coli O157:H7 and Salmonella Enteritidis remained at high levels throughout the incubation period. The results of this study suggest that the contamination of kimchi with E. coli O157:H7, Salmonella Enteritidis, S. aureus, or L. monocytogenes at any stage of production or marketing could pose a potential risk.     

Thermal resistance of Listeria monocytogenes, Salmonella Heidelberg, and Escherichia coli O157:H7 at elevated temperatures

A continuous-flow apparatus was developed to measure thermal resistance (D- and z-values) of microorganisms at temperatures above 65 degrees C. This apparatus was designed to test whether vegetative microorganisms exhibited unusually high thermal resistance that prevented them from being completely eliminated at temperatures applicable to vacuum-steam-vacuum processes (116 to 157 degrees C). The apparatus was composed of a high-pressure liquid chromatography pump, a heating unit, and a cooling unit. It was designed to measure small D-values (<1 s). Three randomly selected organisms, Listeria monocytogenes, Salmonella Heidelberg, and Escherichia coli O157:H7 suspended in deionized water were tested in the continuous-flow apparatus at temperatures ranging from 60 to 80 degrees C. Studies showed that the D-values of these organisms ranged from 0.05 to 20 s. Heating at 80 degrees C was found to be basically the physical limit of the system. Experimental results showed that L. monocytogenes, Salmonella Heidelberg, and E. coli O157:H7 did not exhibit unusual heat resistance. The conditions used in the vacuum-steam-vacuum processes should have completely inactivated organisms such as L. monocytogenes, Salmonella Heidelberg, and E. coli O157:H7 if present on food surfaces. The complete destruction of bacteria during vacuum-steam-vacuum processes might not occur because the surface temperatures never reached those of the steam temperatures and because bacteria might be hidden beneath the surface and was thus never exposed to the destructive effects of the steam.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar against E. coli O157:H7

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (about 10(6) CFUg(-1)) of E. coli O157:H7 and Salmonella enterica serovars Typhimurium on shredded iceberg lettuce and water samples individually, they were stored at 4 degrees C for 14 days and 22 degrees C for 7 days to monitor the growth and survival of pathogens. The results showed that at the end of 4 degrees C storage, populations of two pathogens in lettuce and water decreased approximately 1 log CFUg(-1). However, microbial levels on shredded lettuce increased 3 logs within 3 days at 22 degrees C. Vinegar (acetic acid) had been used to reduce populations of foodborne pathogens in foods; hence, the antimicrobial effect of rice vinegar on the survival of E. coli O157:H7 in inoculated lettuce (10(4) and 10(7) CFUg(-1)) is examined in this study. Results were observed that the treatment of inoculated lettuce (10(7) CFUg(-1)) with commercial vinegar containing 5% acetic acid (pH 3.0) for 5 min would reduce 3 logs population at 25 degrees C. Less than a 1-log decrease in bacterial numbers was recovered during 5 min exposure to 0.5% (pH 3.26) acetic acid.     

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.     

Thermal inactivation of stationary-phase and acid-adapted Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in fruit juices

The heat resistance of stationary-phase and acid-adapted Escherichia coli O157:H7, Salmonella enterica (serotypes Typhimurium, Enteritidis, Gaminara, Rubislaw, and Hartford), and Listeria monocytogenes was evaluated in single-strength apple. orange, and white grape juices adjusted to pH 3.9. The heat resistance increased significantly (P < 0.05) after acid adaptation. Salmonella had an overall lower heat resistance than the other pathogens. Acid-adapted E. coli O157:H7 presented the highest heat resistance in all juices at the temperatures tested, with lower z-values than Salmonella and L. monocytogenes. The heat resistance (D(60 degrees C)-values) of all three pathogens, assessed in tryptic soy broth adjusted to different pH values, increased above pH 4.0. From the results obtained in this study, one example of a treatment that will inactivate 5 logs of vegetative pathogens was calculated as 3 s at 71.1 degrees C (z-value of 5.3 degrees C). Normal processing conditions calculated for hot-filled, shelf-stable juices achieve a lethality in excess of 50,000 D for all three pathogens.     

Calcinated calcium killing of Escherichia coli O157:H7, Salmonella,and Listeria monocytogenes on the surface of tomatoes

This study was conducted to evaluate the efficacy of calcinated calcium, 200 ppm chlorine water (1% active chlorine), and sterile distilled water in killing Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on the surfaces of spot-inoculated tomatoes. Inoculated tomatoes were sprayed with calcinated calcium, chlorinated water, or sterile distilled water (control) and hand rubbed for 30 s. Populations of E coli O157:H7, Salmonella, and L. monocytogenes in the rinse water and in the residual (0.1% peptone) wash solution were determined. Treatment with 200 ppm chlorine and calcinated calcium resulted in 3.40- and 7.85-log10 reductions of E. coli O157:H7, respectively, and 2.07- and 7.36-log10 reductions of Salmonella, respectively. Treatment with 200 ppm chlorine and calcinated calcium reduced L monocytogenes numbers by 2.27 and 7.59 log10 CFU per tomato, respectively. The findings of this study suggest that calcinated calcium could be useful in controlling pathogenic microorganisms in fresh produce.     

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in apple juice with gaseous ozone

This research was initiated to assess the efficacy of gaseous ozone for inactivation Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in apple juice. Juice samples with solids content of 18, 36, and 72 °Brix inoculated with a culture cocktail of three foodborne pathogens were treated with gaseous ozone at a flow rate of 3.0 L/min and an ozone generation rate of 0.10, 0.90, 3.51, and 5.57 g/h for 0.5, 1, 5, and 10 min, respectively. The inactivation kinetics of gaseous ozone on foodborne pathogens conformed to the Weibull model. The time required to achieve a 5 log reduction (t_5d) was estimated using the parameters of the Weibull model. The t_5d increased with increasing solids content of apple juice. The ozone generation rate did not impart a significant effect (p > 0.05) on t_5d. Gaseous ozone is effective at inactivating foodborne pathogens in apple juice but the efficacy is dependent on the solids content of the juice sample.     

Interactions of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes plants cultivated in a gnotobiotic system

The growth and persistence of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes on a diverse range of plant types over extended cultivation periods was studied. When introduced on the seed of carrot, cress, lettuce, radish, spinach and tomato all the pathogens became rapidly established shortly after germination, attaining cell densities of the order of 5.5-6.5 log cfu/g. In general, Es. coli O157:H7 and L. monocytogenes became established and persisted at significantly higher levels on seedlings (9 days post-germination) than Salmonella. Es. coli O157:H7 became internalized in cress, lettuce, radish and spinach seedlings but was not recovered within the tissues of mature plants. Internalization of Salmonella was also observed in lettuce and radish but not cress or spinach seedlings. In contrast, L. monocytogenes did not internalize within seedlings but did persist on the surface of plants throughout the cultivation period. Co-inoculation of isolates recovered from the rhizosphere of plants did not significantly affect the numbers or persistence of human pathogens. The only exception was with Enterobacter cloacae, which reduced Es. coli O157:H7 Ph1 and L. monocytogenes levels by ca. 1 log cfu/g on lettuce. With the bioluminescent phenotype of Es. coli O157:H7 Ph1, it was demonstrated that the human pathogen became established on the roots of growing plants. Scanning electron micrographs of root seedlings suggested that Es. coli O157:H7 Ph1 preferentially colonized the root junctions of seedlings. It is proposed that such colonization sites enhanced the persistence of Es. coli O157:H7 on plants and facilitated internalization within developing seedlings. The results suggest that the risk associated with internalized human pathogens in salad vegetables at harvest is low. Nevertheless, the introduction of human pathogens at an early stage of plant development could enhance their persistence in the rhizosphere. The implications of the study with regards to on-farm food safety initiatives are discussed.     

Combined effect of ultrasound and organic acids to reduce Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on organic fresh lettuce

This study was performed to compare the effectiveness of individual treatments (ultrasound and organic acids) and their combination on reducing foodborne pathogens on organic fresh lettuce. Lettuce leaves were inoculated with a cocktail of three strains each of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes and treated with ultrasound (40 kHz) alone, organic acids (0.3, 0.5, 0.7, 1.0, and 2.0% — malic acid, lactic acid, and citric acid) alone and combined with ultrasound and organic acids for 5 min. For all 3 pathogens, the combined treatment of ultrasound and organic acids resulted in additional 0.8 to 1.0 log reduction compared to individual treatments, without causing significant quality change (color and texture) on lettuce during 7 day storage. The maximum reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were 2.75, 3.18, and 2.87 log CFU/g observed after combined treatment with ultrasound and 2% organic acid for 5 min, respectively. Our results suggest that the combined treatment of ultrasound with organic acids was effective at increasing pathogen reduction compared to individual treatments without significantly affecting quality, and demonstrates its potential as a novel method to increase the microbial safety on organic fresh lettuce.     

Thermal process validation for Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ground turkey and beef products

At 55 to 70 degrees C, thermal inactivation D-values for Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes were 19.05 to 0.038, 43.10 to 0.096, and 33.11 to 0.12 min, respectively, in ground turkey and 21.55 to 0.055, 37.04 to 0.066, and 36.90 to 0.063 min, respectively, in ground beef. The z-values were 5.73, 5.54, and 6.13 degrees C, respectively, in ground turkey and 5.43, 5.74, and 6.01 degrees C, respectively, in ground beef. In both ground turkey and beef, significant (P < 0.05) differences were found in the D-values between E. coli O157:H7 and Salmonella or between E. coli O157:H7 and L. monocytogenes. At 65 to 70 degrees C, D-values for E. coli O157:H7, Salmonella, and L. monocytogenes were also significantly (P < 0.05) different between turkey and beef. The obtained D- and z-values were used in predicting process lethality of the pathogens in ground turkey and beef patties cooked in an air impingement oven and confirmed by inoculation studies for a 7-log (CFU/g) reduction of E. coli O157:H7, Salmonella, and L. monocytogenes.     

Survival of Listeria monocytogenes and Escherichia coli O157:H7 during sauerkraut fermentation

Sauerkraut was produced from shredded cabbage, as is typical in the United States, and from whole head cabbages, which is a traditional process in parts of Eastern Europe. The sauerkraut was inoculated with five strain mixtures of Escherichia coli O157:H7 and Listeria monocytogenes, and the populations of these bacteria, as well as lactic acid bacteria, pH, and titratable acidity, were monitored over the course of fermentation. Fermentation variables were temperature (18 and 22 degrees C) and salt concentration (1.8, 2.25, and 3%). For most of the analyses, the type of cabbage processing was a significant factor, although within cabbage type, neither salt nor fermentation temperature had significant effects. The final pH of the whole-head sauerkraut was lower than the shredded sauerkraut, but the titratable acidity was significantly higher in the shredded sauerkraut. E. coli O157:H7 and L. monocytogenes persisted in the brines for most of the fermentation, although at the end of the fermentations (15 days for shredded, 28 days for whole head), neither pathogen had detectable populations. E. coli populations decreased more rapidly in the shredded sauerkraut even though the pH was higher because of the higher total acidity in the shredded sauerkraut. Acid-tolerant strains of E. coli and L. monocytogenes were isolated from both shredded and whole-head sauerkraut at different salt concentrations and temperatures after 15 days of fermentation and could be detected at 35 days in the wholehead sauerkraut.     

Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties

Thermal inactivation studies were used to determine the D- and z-values of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties. Inoculated meat was packaged in sterile bags, which were immersed in a circulated water bath and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different lengths of time. D- and z-values were determined with a linear regression model. Average D-values at temperatures 55 to 70 degrees C were 27.62 to 0.04 min for E. coli 0157:H7, 67.68 to 0.22 min for Salmonella, and 81.37 to 0.31 min for L. monocytogenes. The z-values were 5.2 degrees C for E. coli O157:H7, 6.0 degrees C for Salmonella, and 6.1 degrees C for L. monocytogenes. The results of this study can be used by food processors to validate their processes and help eliminate pathogenic bacteria associated with chicken-fried beef products.     

Death of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in shelf-stable, dairy-based, pourable salad dressings

The objectives of this study were to determine the death rates of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in three commercially manufactured full-fat ranch salad dressings, three reduced-fat ranch salad dressings, two full-fat blue cheese salad dressings, and two reduced-fat blue cheese salad dressings and to affirm the expectation that these dressings do not support the growth of these pathogens. The respective initial pH values of the four types of shelf-stable, dairy-based, pourable dressings were 2.87 to 3.72, 2.82 to 3.19, 3.08 to 3.87, and 2.83 to 3.49, respectively. Dressings were inoculated with low (2.4 to 2.5 log CFU/g) and high (5.3 to 5.9 log CFU/g) populations of separate five-strain mixtures of each pathogen and stored at 25 degrees C for up to 15 days. Regardless of the initial inoculum population, all test pathogens rapidly died in all salad dressings. Salmonella was undetectable by enrichment (<1 CFU/25-ml sample in three replicate trials) in all salad dressings within 1 day, and E. coli O157:H7 and L. monocytogenes were reduced to undetectable levels by enrichment between 1 and 8 days and 2 and 8 days, respectively. E. coli O157:H7 was not detected in 4 of the 10 salad dressings stored for 2 or more days and 9 of the 10 dressings stored for 6 or more days after inoculation. L. monocytogenes was detected in 9 of the 10 salad dressings stored for 3 days but in only one dressing, by enrichment, at 6 days, indicating that it had the highest tolerance among the three pathogens to the acidic environment imposed by the dressings. Overall, the type of dressing (i.e., ranch versus blue cheese) and level of fat in the dressings did not have a marked effect on the rate of inactivation of pathogens. Total counts and populations of lactic acid bacteria and yeasts and molds remained low or undetectable (<1.0 log CFU/ml) throughout the 15-day storage period. Based on these observations, shelf-stable, dairy-based, pourable ranch and blue cheese salad dressings manufactured by three companies and stored at 25 degrees C do not support the growth of Salmonella, E. coli O157:H7, and L. monocytogenes and should not be considered as potentially hazardous foods (time-temperature control for safety foods) as defined by the U.S. Food and Drug Administration Food Code.     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

Survival of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated walnut kernels during storage

The survival of single strains or cocktails of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on walnut kernels. Kernels were separately inoculated with an aqueous preparation of the pathogens at 3 to 10 log CFU/g, dried for 7 days, and then stored at 23°C for 3 weeks to more than 1 year. A rapid decrease of 1 to greater than 4 log CFU/g was observed as the inoculum dried. In some cases, the time of storage at 23°C did not influence bacterial levels, and in other cases the calculated rates of decline for Salmonella (0.05 to 0.35 log CFU/g per month) and E. coli O157:H7 (0.21 to 0.86 log CFU/g per month) overlapped and were both lower than the range of calculated declines for L. monocytogenes (1.1 to 1.3 log CFU/g per month). In a separate study, kernels were inoculated with Salmonella Enteritidis PT 30 at 4.2 log CFU/g, dried (final level, 1.9 log CFU/g), and stored at -20, 4, and 23°C for 1 year. Salmonella Enteritidis PT 30 declined at a rate of 0.10 log CFU/g per month at 23°C; storage time did not significantly affect levels on kernels stored at -20 or 4°C. These results indicate the long-term viability of Salmonella, E. coli O157:H7, and L. monocytogenes on walnut kernels and support inclusion of these organisms in hazard assessments.     

Cross-contamination of lettuce with Escherichia coli O157:H7

Contamination of produce by bacterial pathogens is an increasingly recognized problem. In March 1999, 72 patrons of a Nebraska restaurant were infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7, and shredded iceberg lettuce was implicated as the food source. We simulated the restaurant's lettuce preparation procedure to determine the extent of possible EHEC cross-contamination and growth during handling. EHEC inoculation experiments were conducted to simulate the restaurant's cutting procedure and the subsequent storage of shredded lettuce in water in the refrigerator. All lettuce pieces were contaminated after 24 h of storage in inoculated water (2 x 10(9) CFU of EHEC per 3 liters of water) at room temperature or at 4 degrees C; EHEC levels associated with lettuce increased by > 1.5 logs on the second day of storage at 4 degrees C. All lettuce pieces were contaminated after 24 h of storage in water containing one inoculated lettuce piece (approximately 10(5) CFU of EHEC per lettuce piece) at both temperatures. The mixing of one inoculated dry lettuce piece with a large volume of dry lettuce, followed by storage at 4 degrees C or 25 degrees C for 20 h resulted in 100% contamination of the leaves tested. Microcolonies were observed on lettuce stored at 25 degrees C, while only single cells were seen on leaves stored at 4 degrees C, suggesting that bacterial growth had occurred at room temperature. Three water washes did not significantly decrease the number of contaminated leaves. Washing with 2,000 mg of calcium hypochlorite per liter significantly reduced the number of contaminated pieces but did not eliminate contamination on large numbers of leaves. Temperature abuse during storage at 25 degrees C for 20 h decreased the effectiveness of the calcium hypochlorite treatment, most likely because of bacterial growth during the storage period. These data indicate that storage of cut lettuce in water is not advisable and that strict attention must be paid to temperature control during the storage of cut lettuce.