ABSTRACT MATERIALS AND METHODS PETER J. TAORMINA AND LARRY R. BEUCHAT*

Transcription

1 850 Journal of Food Protection, Vol. 62, No. 8, 1999, Pages Copyright, International Association of Milk, Food and Environmental Sanitarians Behavior of Enterohemorrhagic Escherichia coli O157:H7 on Alfalfa Sprouts during the Sprouting Process as Influenced by Treatments with Various s PETER J. TAORMINA AND LARRY R. BEUCHAT* Center for Food Safety and Quality Enhancement, Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, Georgia , USA MS : Received 4 September 1998/Accepted 20 November 1998 ABSTRACT The behavior of Escherichia coli O157:H7 on alfalfa seeds subjected to conditions similar to those used commercially to grow and market sprouts as it is affected by applications of NaOCl, Ca(OCl) 2, acidified NaClO 2, acidified ClO 2,Na 3 PO 4, Vegi-Clean, Tsunami, Vortexx, or H 2 O 2 at various stages of the sprouting process was determined. Application of ppm of NaOCl, 200 and ppm of Ca(OCl) 2, 500 ppm of acidified ClO 2, 10,000 ppm of Vegi-Clean, ppm of Tsunami, or 40 and ppm of Vortexx to germinated seeds significantly reduced the population of E. coli O157:H7. With the exception of acidified NaOCl 2 at 1,200 ppm, spray applications of these chemicals did not significantly reduce populations or control the growth of E. coli O157:H7 on alfalfa sprouts during the sprouting process. Populations of E. coli on alfalfa sprouts peaked at 6 to 7 log 10 CFU/g 48 h after initiation of the sprouting process and remained stable despite further spraying with chemicals. The population of E. coli O157:H7 on sprouts as they entered cold storage at 9 2 C remained essentially unchanged for up to 6 days. None of the chemical treatments evaluated was able to eliminate or satisfactorily reduce E. coli O157:H7 on alfalfa seeds and sprouts. Observations on the ability of E. coli O157:H7 to grow during production of alfalfa sprouts not subjected to chemical treatments are similar to those from a previous study in our laboratory on the behavior of Salmonella Stanley. Our results do not reveal a chemical treatment method to eliminate the pathogen from alfalfa sprouts. We have demonstrated that currently recommended procedures for sanitizing alfalfa seeds fail to eliminate E. coli O157:H7 and that the pathogen can grow to populations exceeding 7 log 10 CFU/g of sprouts produced using techniques not dissimilar to those used in the sprout industry. Ground beef has been the food most often associated with outbreaks of Escherichia coli O157:H7 infection (6, 8). However, outbreaks have been linked to unpasteurized apple cider (1, 4, 20) and leaf lettuce (2, 3). In 1996, white radish sprouts were implicated in the world s largest outbreak of E. coli O157:H7 infection in Japan (9, 15). Alfalfa sprouts have been linked to numerous outbreaks of salmonellosis in the United States in recent years (7, 14, 16, 18, 23). In the summer of 1997, outbreaks of E. coli O157:H7 infections in Michigan and Virginia were linked to alfalfa sprouts (5). In the fall of the same year, outbreaks of Salmonella Senftenberg infections in Northern California were associated with the consumption of an alfalfa and clover sprout mixture (19). A cluster of E. coli O157:NM infections in California also associated with eating an alfalfa and clover sprout mixture was also reported in the summer of 1998 (19). In several of these cases, alfalfa seeds used for sprouting were implicated as the source of the pathogen. Alfalfa seeds are a raw agricultural commodity that may become contaminated with pathogens such as Salmonella or E. coli O157:H7 en route from the farm to the sprouting facility. The sprouting process provides a warm * Author for correspondence. Tel: ; Fax: ; lbeucha@cfsqe.griffin.peachnet.edu. and moist environment favorable for the growth of bacteria, including human pathogens that may be present on the seeds. This study was designed to evaluate the behavior of E. coli O157:H7 on alfalfa seeds subjected to conditions similar to those used commercially to grow and market sprouts as it is affected by applications of chemical treatments at various stages of the sprouting process. Survival of the pathogen on mature sprouts stored at refrigeration temperatures was also studied. MATERIALS AND METHODS Strains and preparation of inoculum. Five enterohemorrhagic strains of E. coli O157:H7 were studied: strain 932 (human isolate), strain 994 (salami isolate), strain E0018 (calf fecal isolate), strain H1730 (human isolate associated with consuming lettuce), and strain F4546 (human isolate associated with consuming alfalfa sprouts). Laboratory stock cultures were activated by transferring loop inocula into 10 ml of tryptic soy broth (TSB, ph 7.3) (Difco Laboratories, Detroit, Mich.), then adapting cells to grow in TSB containing 50 g/ml of nalidixic acid (TSBN) (Sigma Co., St. Louis, Mo.). Three successive 24-h transfers were made in TSBN prior to use as inocula for experiments. Procedure for inoculating alfalfa seeds. Alfalfa seeds were obtained from Caudill Seed Co., Louisville, Ky., and had a germination percentage of approximately 91%. Sterile deionized water (1 liter) was combined with 30 ml of a 24-h TSBN culture

2 J. Food Prot., Vol. 62, No. 8 E. COLI O157:H7 ON ALFALFA SEEDS 851 TABLE 1. Overall quality scores for appearance of mature alfalfa sprouts dipped in various chemicals and stored for up to 10 days at 9 2 C Sprout quality a 1 day 3 days 6 days 8 days 10 days Tap water b NaOCl 200 Ca(OCl) Acidified NaOCl ,200 Na 3 PO 4 40, ,000 Tsunami 40 Vortexx 40 H 2 O 2 10,000 50, a Quality was ranked on a 5-point scale: 5, excellent quality; 4, good quality; 3, average quality; 2, poor quality; 1, inedible. Values are averaged scores of four judges. b Control. TABLE 2. Overall quality scores for appearance of alfalfa sprouts dipped in various chemicals, rinsed in tap water, and stored for up to 10 days at 9 2 C Sprout quality a 1 day 3 days 6 days 8 days 10 days Tap water b NaOCl 200 Ca(OCl) Acidified NaOCl ,200 Na 3 PO 4 40, ,000 Tsunami 40 Vortexx 40 H 2 O 2 10,000 50, a Quality was ranked on a 5-point scale: 5, excellent quality; 4, good quality; 3, average quality; 2, poor quality; 1, inedible. Values are averaged scores of four judges. b Control. containing 6 ml of each of five E. coli O157:H7 strains. Dry seed (1 kg) was combined with the cell suspension and gently mixed for 1 min. The suspension was decanted, and seeds were placed on wire screens lined with cheesecloth to dry at 23 2 C under a laminar flow hood for 24 h. Seeds were then placed in plastic bags and held at 5 C until used in sprout production experiments. Preparation of chemical treatment solutions. Selection of chemical treatment solutions evaluated in this experiment was based on certain criteria. Besides either meeting or having potential to meet criteria as direct-contact disinfectants for produce, chemicals were selected at concentrations that were effective against E. coli O157:H7 on alfalfa seeds in previous experiments. Finally, a panel of four people evaluated the overall appearance of mature alfalfa sprouts that had been dipped in chemical solutions and rinsed in tap water or not rinsed. The panel rated the appearance of the alfalfa sprouts during 10 days of storage at 9 2 C and assigned numerical values on a 1 to 5 scale (1 inedible, 2 poor quality, 3 average quality, 4 good quality, and 5 excellent quality). The following chemical treatments were evaluated for their efficacy in killing E. coli O157:H7 on alfalfa seeds and sprouts: NaOCl (Aldrich, Milwaukee, Wis.), 0, 200, and g of active chlorine per ml of 0.05 M potassium phosphate buffer (ph 6.8); Ca(OCl) 2 (J. T. Baker Inc., Phillipsburg, N.J.), 0, 200, and g of active chlorine per ml of 0.05 M potassium phosphate buffer (ph 6.8); acidified NaClO 2 (Alcide Corp., Redmond, Wash.), 0, 500, and 1,200 g of NaClO 2 per ml; USS-1400 (acidified ClO 2 ) (Universal Sanitizers and Supplies, Knoxville, Tenn.), 0, 100, and 500 g of free or bound ClO 2 per ml of distilled water; hydrogen peroxide (H 2 O 2 ) (VWR, West Chester, Pa.), 0 and 1% (vol/vol) in deionized water; trisodium phosphate (Na 3 PO 4 ) (Rhone-Poulenc Food Ingredients, Cranberry, N.J.), 0 and 1% (vol/vol) in deionized water; Vegi-Clean (Microcide, Inc., Detroit, Mich.), 0 and 1% (wt/ vol) in deionized water; Tsunami (active oxygen solution) (Ecolab, Mendota Heights, Minn.), 0, 40, and /ml of deionized water; and Vortexx (active oxygen solution) (Ecolab), 0, 40, and /ml of deionized water. Sterile tap water containing no added chemicals served as a control. The temperature of seeds and sprouts and treatment solutions was 22 2 C. Free chlorine in Ca(OCl) 2 and NaOCl solutions was determined with a chlorine test kit (Hach Co., Ames, Iowa), which is approved by the U.S. Environmental Protection Agency. s of USS-1400 acidified ClO 2 solutions were confirmed with a stabilized ClO 2 titration kit (Universal Sanitizers and Supplies). Seed germination, sprouting, and storage. Inoculated alfalfa seeds were combined with uninoculated seeds in a 3:1 (wt/ wt) ratio to achieve an E. coli O157:H7 population of 1.94 to 6 log 10 CFU/g. Seeds (150 g) were weighed separately into plastic vegetable bags ( cm) (DowBrands, Indianapolis, Ind.) perforated with holes smaller than the size of alfalfa seeds. Seeds were presoaked by immersion into 1 liter of chemical treatment solution or water (control) for 20 min with occasional agitation. the presoak, seeds were drained for 2 min and then immersed in 1 liter of sterile tap water for 1 h. Seeds were again drained and aseptically transferred into plastic boxes ( cm) with drainage holes of approximately 0.5-cm diameter in the bottom. Soaked seeds were spread evenly (approximately 0.7-cm thick) in boxes that were then covered with a sheet of perforated plastic fabricated from vegetable bags (DowBrands) and placed in an incubator at 21 C to germinate. 24 h, boxes of germinated seeds were placed under fluorescent light for an

3 852 TAORMINA AND BEUCHAT J. Food Prot., Vol. 62, No. 8 TABLE 3. Effects of spray application of chemical sanitizers on the population of E. coli O157:H7 on alfalfa sprouts during the sprouting process Treatment Initial Presoak Rinse d Germination b e Water NaOCl Ca(OCl) 2 Acidified NaClO ,200 F 2.15 B B 2.47 A F 3 A D 2.62 A FG 2.10 B DE 1.94 B ABC 2.12 B E 2.63 B B 1 B G 8 C D 3.20 A G 1.62 DC E 1.24 D BC 0 D E 6 A B 2.71 A FG 2.28 A D 8 A F 2.40 A E 7 B C 8 B AB 5.50 A A 5.31 AB ABC 4.93 AB A 5.32 AB ABC 4.86 B BC 9 C ABC 6 C ABC 5.30 A A 4.66 AB DE 4 BC BC 4.62 B E 4 C CD 2.6 D C 3 E ABCD 5.00 A A 4.95 A ABC 4.95 A A 5.37 A AB 5.00 A A 7 A A 2.92 B a Ten grams of alfalfa seeds was analyzed before and after a 20-min presoak in treatment solution (initial and presoak population) and after a 60-min rinse in sterile tap water; 20 g of sprouts were analyzed at stages of germination and sprouting before and after spray application of chemicals. Values in the same row (within the same treatment) that are preceded by the same letter are not significantly different. Values in the same column (within the same sampling time) that are followed by the same letter are not significantly different. b Seeds were germinated 24 h at 21 C. c Sprouting was carried out at 21 C. d Population on sprouts before application of chemicals. e Population on sprouts immediately after application of chemicals and rinsing with sterile tap water. f Mature sprouts were stored in 9 2 C. additional 72 h. Germinating alfalfa seeds and sprouts in each box were watered every 4 to 6 h by spraying 35 to 40 ml of sterile tap water evenly onto the surface. Mature sprouts (72 h postgermination) were removed from the sprouting chamber, weighed (100 g) into clear plastic display trays with lids (base dimensions, 8 8 cm; height, 6.5 cm; top, cm), and placed into cold storage (9 2 C) for up to 6 days. Procedures for determining populations of E. coli O157: H7 and applying chemical solutions. Alfalfa seeds (10 g) and 20 ml of sterile 0.1% peptone water were combined in a stomacher bag and pummeled at medium speed for 30 s using a stomacher (model, Seward Medical Limited, London, UK). Undiluted peptone wash water (0.25 ml) was surface plated in quadruplicate on sorbitol MacConkey agar (SMA) (Oxoid, Basingstoke, Hampshire, UK) supplemented with 50 /ml of nalidixic acid (SMAN) or surface plated (0.1 ml) in duplicate on SMAN. This sampling procedure was performed on seeds before and after the 20-min presoak step and after soaking for 1 h in sterile tap water. In addition to presoaking seeds in chemical solutions for 20 min, 40 to 45 ml of test chemicals and water (control) was sprayed evenly onto sprouts in each box after 24 h of germination and 24, 48, and 72 h after the sprouts were placed under fluorescent light. solutions sprayed onto sprouts were rinsed 5 min after application by spraying 20 to 25 ml of sterile tap water evenly. Samples of sprouts (20 g) were removed from each box and analyzed for populations of E. coli O157:H7 prior to and immediately after spray application of chemical solutions. Samples were combined with 20 ml of 0.1% peptone water in a plastic bag and massaged gently, but thoroughly, by hand for 30 s before surface plating quadruplicate 0.25-ml samples and duplicate undiluted or serially diluted 0.1-ml samples on SMAN. Sampling of sprouts stored at 9 2 C was similar, except 100 g of sprouts was combined with 100 ml of 0.1% peptone water. Presumptive colonies of E. coli O157:H7 formed on SMAN after 24 h of incubation at 37 C were counted. Three presumptive colonies from randomly selected plates containing 30 to 300 colonies were streaked onto tryptic soy agar (TSA) (Difco). Isolated colonies formed on TSA after incubation at 37 C for 24 h were confirmed by latex agglutination reaction (Unipath-Oxoid, Columbia, Md.) and API 20E diagnostic kits (biomérieux Vitek, Inc., Hazelwood, Mo.). Statistical analysis. Three replicate trials for each treatment were conducted. Data from each treatment were subjected to the Statistical Analysis System (SAS; SAS Institute, Cary, N.C.) for analysis of variance (ANOVA) and Duncan s multiple range tests to determine significant differences within a given treatment over various sampling times. Data generated from three separate experiments having three separate controls were independently subjected to the SAS for ANOVA and Duncan s multiple range tests to determine significant differences at a given sampling time. Each value presented represents a mean of six values (duplicate values from each sample from three replicate trials). RESULTS In a previous study (21), we evaluated various concentrations of several chemicals for their efficacy in killing E. coli O157:H7 on alfalfa seeds. While some chemicals showed promise as disinfectants, their effects on sensory qualities of mature alfalfa sprouts were not determined. Preliminary studies were therefore carried out to determine if various concentrations of chemicals adversely affected appearance and color of mature sprouts before selecting concentrations of treatment solutions to apply to sprouts during their production. Results of the subjective evaluation of visual appearance of mature alfalfa sprouts that were dipped in chemical solutions and stored for up to 10 days at 9 2 C are listed in Table 1. Results of similar evaluations of sprouts that had been dipped in chemicals and then rinsed with tap water are listed in Table 2. Values presented are means obtained from evaluations by four judges. Rinsing sprouts after application of chemical treatments clearly helped to maintain overall appearance. solutions at concentrations that showed little or no detrimental effects to appearance of alfalfa sprouts were selected for testing

4 J. Food Prot., Vol. 62, No. 8 E. COLI O157:H7 ON ALFALFA SEEDS 853 TABLE 3. Extended Sprouting (48 h) Sprouting (72 h) Storage f 24 h 96 h 144 h D 3 ABC A 4.90 AB E 3.91 BC C 1 BC A 5.30 A AB 7 C ABC 0 D BCD 4.78 A A 5.27 A BCD 4.56 A ABC 4.84 A AB 5.02 A BC 3.10 B ABC 0 B ABC 5.15 A A 4.91 AB CDE 4 AB ABC 4.83 AB ED 7 BC BC 3.15 CD AB 2.75 D CD 4.56 A A 5.23 A BCD 4.56 A AB 5.10 A ABC 4.83 A ABC 3 B ABC 8 C ABCD 4.96 A A 5.32 A DE 4.17 AB AB 5.00 A DC 6 A BC 0 BC ABC 4 C A 5.51 A A 5.08 A A 5.20 A A 5.45 A AB 5.20 A AB 0 B ABC 2.41 C ABCD 4.99 A A 4.87 A AB 5.07 A AB 5.00 A AB 5.26 A BC 2 B ABC 2.21 B ABCD 4.90 A A 4.61 A BCD 4.60 A ABC 4.85 A BC 4.74 A BC 3.26 B ABC 7 C effectiveness against E. coli O157:H7 on alfalfa sprouts during their production. Two additional chemicals (acidified ClO 2 and Vegi-Clean) were also tested. Evaluation of the application of various concentrations of chemicals in reducing the population of E. coli O157: H7 on alfalfa seeds and sprouts during the sprouting process was performed in three separate sets of experiments. Each of the three sprouting trials included seeds and sprouts that were subjected to treatments with a water control and a -ppm NaOCl treatment recommended by the U.S. Food and Drug Administration (22). Presoaking alfalfa seeds in ppm of NaOCl, 1,0 ppm of Ca(OCl) 2,or 60,000 ppm of H 2 O 2 in at least a 5:1 water-to-seed ratio for 10 min is recommended for alfalfa seeds destined for sprout production (11). The results of the first sprouting trial are summarized in Table 3. The initial population of E. coli O157:H7 on alfalfa seeds used in this set of experiments ranged from 1.94 to 2.62 log 10 CFU/g. Populations of E. coli O157:H7 recovered from seeds following a 20- min presoak in the chemical treatment solutions and a 1-h soak in sterile tap water were not significantly different from the initial population. Seeds treated with water contained significantly higher populations compared with those detected before presoak and rinse treatments. The population of E. coli O157:H7 on seeds after a 24-h germination period, but prior to spray application of chemicals, was significantly higher on seeds presoaked in all treatments except 1,200 ppm of acidified NaClO 2. Application of 200 or ppm of Ca(OCl) 2 and ppm of NaOCl to germinated seeds significantly reduced the population of E. coli O157:H7. In most cases further spray applications of chemicals did not significantly reduce populations of E. coli O157:H7 on alfalfa sprouts during the sprouting process. Generally, highest numbers of E. coli O157:H7 were recovered from sprouts at the end of the sprout production stage, i.e., 96 h after the initiation of sprout production. Only treatment with 1,200 ppm of acidified NaOCl 2 controlled growth of the pathogen during sprout growth; the population of E. coli O157:H7 at the last sample time (144 h of storage, 240 h after seed rinse) was not significantly different from the initial population on dry seeds. Results from the second set of experiments are listed in Table 4. There were no significant differences in the initial populations of E. coli O157:H7 recovered from dry alfalfa seeds prior to application of treatments. Seeds presoaked for 20 min in 500 ppm of acidified ClO 2 resulted in the greatest reduction in population of the pathogen. Populations of E. coli O157:H7 recovered from seeds presoaked for 20 min in water, 10,000 ppm of Na 3 PO 4, or 10,000 ppm of Vegi-Clean and then rinsed in sterile tap water for 1 h were significantly higher than initial populations. the 24-h germination period, populations of the pathogen increased by 2.4 to log 10 CFU/g of seeds. Spray application of chemicals to germinated seeds revealed that only 500 ppm of acidified ClO 2 and 10,000 ppm of Vegi-Clean significantly reduced the number of E. coli O157:H7. During the sprouting process, populations of E. coli O157:H7 recovered from sprouts following spray application of chemicals was not significantly lower than populations recovered before application, except for 100 ppm of acidified ClO 2 at 48-h sprouting (72 h total). The population of E. coli O157:H7 on sprouts as they entered cold storage ranged from 4.92 to 6.53 log 10 CFU/g and remained stable for up to 6 days. No chemical treatment was consistent in significantly reducing numbers of E. coli O157: H7 on sprouts when compared with numbers recovered from water-treated sprouts, and there was no significant difference between various treatments after 6 days of cold storage. Results from the third set of chemical treatments on alfalfa sprouts are listed in Table 5. Initial populations of E. coli O157:H7 on dry alfalfa seeds used in this sprouting trial ranged from 2.62 to 4 log 10 CFU/g, and populations were significantly reduced only on rinsed seeds that had been subjected to a 20-min presoak in ppm of Tsunami. A sharp increase in population of E. coli O157:H7 in all samples was noted after the 24-h germination period. Spray application of ppm of Tsunami or 40 and ppm of Vortexx significantly reduced the population on germinated seeds, although reduction was approximately 1 log at best. Populations peaked after 24 h of sprouting and remained stable despite further spraying with chemicals. The population of E. coli O157:H7 on sprouts stored at 9 2 C remained essentially unchanged. DISCUSSION In each of the three sprout production trials, there were cases when the population of E. coli O157:H7 recovered from soaked seeds was greater and sometimes significantly higher than the initial population or the population on pre-

5 854 TAORMINA AND BEUCHAT J. Food Prot., Vol. 62, No. 8 TABLE 4. Effects of spray application of chemical sanitizers on the population of E. coli O157:H7 on alfalfa sprouts during the sprouting process Initial Presoak Rinse d Germination b e Water NaOCl Acidified NaClO 2 Na 3 PO 4 Vegi-Clean ,000 10,000 H 1 A D 2.43 A E 2.23 A D 6 A D 2.19 A E 9 A G 5 A D 2.77 A D 3.11 A E 1.18 B CD 7 A D 2.92 A F 3.19 A D 9 A DE 2.94 A E 7 B C 8 A D 3.11 A ABCD 6.25 AB ABC 5.29 B ABC 5.72 AB B 4 C AB 6.41 A B 6.25 AB ABCD 6.26 A BC 5.05 B BC 5.32 B C 3.49 C AB 5.76 AB C 5.58 AB BCD 6.17 A A 6.16 A AB 6.10 A A 6.18 A AB 6.15 A A 6.86 A a Ten grams of alfalfa seeds was analyzed before and after a 20-min presoak in treatment solution (initial and presoak population) and after a 60-min rinse in sterile tap water; 20 g of sprouts were analyzed at stages of germination and sprouting before and after spray application of chemicals. Values in the same row (within the same treatment) that are preceded by the same letter are not significantly different. Values in the same column (within the same sampling time) that are followed by the same letter are not significantly different. b Seeds were germinated 24 h at 21 C. c Sprouting was carried out at 21 C. d Population on sprouts before application of chemicals. e Population on sprouts immediately after application of chemicals and rinsing with sterile tap water. f Mature sprouts were stored in 9 2 C. soaked seeds. This occurred with control as well as chemically treated seeds and can be attributed to the seeds imbibing water and releasing cells previously trapped in the cracks and crevices of the seed. In a few instances, the population of E. coli O157:H7 recovered from alfalfa sprouts before chemical spray application was lower than the population recovered after application. Care was taken to spray all samples evenly and thoroughly, and since this seemed to be a random occurrence that was not repeated within a given treatment, it is possible that clusters of cells of E. coli O157: H7 developed in pockets of sprouts or perhaps within the root mat and were protected from the chemicals. Acidified NaClO 2, at 1,200 ppm, performed better than the other chemicals evaluated during that particular sprouting trial and, based on its ability to control the growth of E. coli O157:H7, was superior to all other treatments evaluated. However, none of the chemical treatments evaluated was able to eliminate or dramatically reduce numbers of E. coli O157:H7 on alfalfa seeds and sprouts. Jaquette et al. (13) showed that populations of Salmonella Stanley present on alfalfa seeds at the initiation of the sprouting process can survive and grow on sprouts exposed to commercially used production and handling practices. Hara-Kudo et al. (10) demonstrated that radish sprouts can become heavily TABLE 5. Effects of spray applications of chemical sanitizers on the population of E. coli O157:H7 on alfalfa sprouts during the sprouting process Initial Presoak Rinse d Germination b e Water NaOCl Tsunami Vortexx H 2 O ,000 F 2.71 B CD 2 B C 2 B F 4 A D 2.62 B C 2.64 B C 2.62 B F 2.94 B CD 3.42 A C 1 BC H 1.93 D D 2.65 BC C 2.64 BC C 2.21 CD E 3 A D 2.62 B C 6 AB G 2.65 B C 3.22 A C 2.93 AB C 2.68 B D 6.25 BC BC 4.92 E B 6.32 ABC D 6.16 C A 6.69 A A 6.66 AB C 5.50 D D 6.14 AB CD 4.72 E B 6.04 ABC E 5.59 DC B 6.20 A B 5.64 BCD B 5.45 D A 7.36 A AB 6.93 A A 7.16 A A 6.95 A A 6.98 A A 6.96 A A 6. A A 7.35 A AB 6.72 A AB 6.56 A ABC 6.61 A A 6.94 A A 6.70 A A 6.99 A a Ten grams of alfalfa seeds was analyzed before and after a 20-min presoak in treatment solution (initial and presoak population) and after a 60-min rinse in sterile tap water; 20 g of sprouts were analyzed at stages of germination and sprouting before and after spray application of chemicals. Values in the same row (within the same treatment) that are preceded by the same letter are not significantly different. Values in the same column (within the same sampling time) that are followed by the same letter are not significantly different. b Seeds were germinated 24 h at 21 C. c Sprouting was carried out at 21 C. d Population on sprouts before application of chemicals. e Population on sprouts immediately after application of chemicals and rinsing with sterile tap water. f Mature sprouts were stored in 9 2 C.

6 J. Food Prot., Vol. 62, No. 8 E. COLI O157:H7 ON ALFALFA SEEDS 855 TABLE 4. Extended Sprouting (48 h) Sprouting (72 h) Storage f 24 h 96 h 144 h A 6.54 A ABC 5.68 A ABC 5.60 A A 5.55 A B 5.64 A AB 6.70 A ABC 6.33 A A 6.11 A A 6.27 A A 6.04 A AB 6.39 A AB 6.59 A AB 6.44 A ABC 5.51 B C 5.07 B A 5.57 B AB 6.52 A A 6.84 A CDE 5.99 A BC 5.13 B AB 6.16 A A 6.09 A AB 6.32 A AB 6.58 A BCD 6.12 AB BC 5.04 B ABC 5.81 AB A 5.35 AB A 6.58 A AB 6.52 A DE 5.97 AB C 4.92 C AB 6.08 AB A 5.66 BC AB 6.38 AB AB 6.53 A DE 5.91 AB AB 5.87 AB A 6.20 AB A 5.68 B AB 6.00 AB AB 6.34 A E 5.69 A ABC 5. A AB 5.99 A A 5.58 A AB 5.93 A AB 6.30 A contaminated with E. coli O157:H7 when seeds had been soaked prior to sprouting in water inoculated with the pathogen. Rice seeds inoculated with Bacillus cereus and Listeria can support the growth of these pathogens without any antagonistic effect from the background flora during germination (17). Our observations and those of others clearly demonstrate that a wide range of seed sprouts can support the growth of pathogens known to cause human illness. Our observations on the ability of E. coli O157:H7 to grow during production of alfalfa sprouts not subjected to chemical treatments are similar to observations of the behavior of Salmonella Stanley on alfalfa sprouts (13). Our results do not reveal a chemical treatment method to eliminate the pathogen from sprouts. In a study on the behavior of E. coli O157:H7 on radish sprouts, Itoh et al. (12) were unable to kill the pathogen by treating the outer surface of the hypocotyl with HgCl 2. Through the use of immunofluorescence microscopy and scanning electron microscopy, this group observed the presence of viable cells of E. coli O157:H7 in the inner tissues and stomata of cotyledons of radish sprouts grown from inoculated seeds. The same phenomenon may be occurring with alfalfa sprouts. Further experiments using this technology to view actual cells of E. coli O157:H7 on or within alfalfa sprouts are warranted. In our study, presumptions cannot be made as to the precise location of cells of E. coli O157:H7 in alfalfa seeds and sprouts. Wherever cells may be lodged, reductions of 2 logs of E. coli O157:H7 per g of sprouts as a result of treatment with various chemicals has little practical significance when the number of surviving cells exceeds that necessary to cause infection. We have demonstrated that recommended and experimental disinfection procedures for seeds fail to eliminate E. coli O157:H7 and that the pathogen grows to populations exceeding 7 log 10 CFU/g of sprouts produced using techniques not dissimilar to those used in the sprout industry. REFERENCES 1. Besser, R. E., S. M. Lett, J. T. Weber, M. P. Doyle, T. J. Barrett, J. G. Wells, and P. M. Griffin An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh pressed apple cider. JAMA 269: TABLE 5. Extended Sprouting (48 h) Sprouting (72 h) Storage f 24 h 96 h 144 h AB 7.19 A AB 6.51 A A 6.82 A ABC 6.78 A A 6.99 A A 6. A A 6.61 A AB 7.02 A A 6.66 A A 6.88 A AB 6.86 A A 7.01 A A 6.78 A A 6.70 A AB 7.07 A B 6.12 B A 6.85 AB AB 6.91 A A 6.87 AB A 6. AB A 6.65 AB BC 6.87 A B 5.87 B AB 6.64 AB ABC 6.75 AB A 6.99 A A 6.64 AB A 6.63 AB BC 6.87 A B 6.27 B A 6.75 AB ABC 6.82 AB A 6.91 A A 6.76 AB A 6.82 AB DC 6.54 AB B 6.02 B AB 6.55 AB BCD 6.47 AB A 6.95 A A 6.66 A A 6.62 A DC 6.50 AB B 6.01 B B 6.36 AB DC 6.40 AB A 6. A A 6.62 A A 6.53 AB

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