Combined effects of chemical, heat and ultrasound treatments to kill Salmonella and Escherichia coli O157:H7 on alfalfa seeds
Larry R. Beuchattul Center for Food Safety and Department of food Science and Technology, University of Georgia, 1109 Experiment St., Griffin, GA 30223–1797, USA (e-mail: lbeuchat@cfs. griffin.peachnet.edu).
Aims: To determine the effectiveness of combined treatments with chemicals, heat and ultrasound in killing or removing Salmonella and Escherichia coli O157:H7 on alfalfa seeds intended for sprout production.
Methods and Results: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 were treated with ultrasound (38·5–40·5 kHz) in solutions containing 1% Ca(OH)2, 1% Tween 80, 1% Ca(OH)2 plus 1% Tween 80, 160 μg ml−1 Tsunami® 200 and 0·5% Fit® at 23 and 55°C for 2 and 5 min. Highest reductions were in chemical solutions at 55°C, but seed viability was also reduced compared with treatment at 23°C. Inactivation of Salmonella and E. coli O157:H7 was generally enhanced by simultaneous treatments with ultrasound, chemicals and heat.
Conclusions: Ultrasound treatment, in combination with chemicals and heat, had a modest enhancing effect on the effectiveness of chemicals in killing or removing pathogens on alfalfa seeds. Overall, treatment with 1% Ca(OH)2 was most effective in killing Salmonella and E. coli O157:H7.
Significance and Impact of the Study: The use of 1% Ca(OH)2 instead of 20 000 μg ml−1 chlorine, which is currently recommended as a sanitizer for seeds intended for sprout production in the US, should be considered. Ultrasound treatment of alfalfa seeds containing Salmonella or E. coli O157:H7, in combination with chemical treatment, contributes to achieving greater reductions in populations of these pathogens, thereby reducing the risk of contamination and the presence of pathogens in sprouts produced from these seeds.
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Vegetable seed sprouts have been implicated in several outbreaks of salmonellosis and Escherichia coli O157:H7 infections (National Advisory Committee on Microbiological Criteria for Foods 1999; Taormina et al. 1999). The majority of these outbreaks have involved alfalfa sprouts but mung bean, clover, radish, mustard and cress sprouts have also been associated with infections. The largest outbreak, involving more than 6000 culture-confirmed cases of E. coli O157:H7 infection, was associated with radish sprouts in Japan (Guiterrez 1997; Ministry of Health and Welfare of Japan 1997). Salmonella and E. coli O157:H7 can grow to populations of up to 107 cfu g−1 of alfalfa sprouts during production and retain viability during storage at refrigeration temperature (Jaquette et al. 1996; Lang et al. 2000; Stewart et al. 2001; Taormina and Beuchat 1999a).
The source of human pathogens on sprouts implicated in many outbreaks of infections is thought to be from seeds rather than contamination of sprouts during or after production. Numerous aqueous chemical treatments have been investigated for their effectiveness in killing or removing Salmonella and E. coli O157:H7 on alfalfa seeds (Jaquette et al. 1996; Beuchat 1997; Taormina and Beuchat 1999b; Lang et al. 2000; Weissinger and Beuchat 2000; Beuchat and Scouten 2001; Beuchat et al. 2001). Hypochlorite, chlorine dioxide, organic acids, hydrogen peroxide, ethanol, trisodium phosphate, calcium hydroxide and commercial formulations containing antimicrobials have exhibited a range of efficacies in killing these pathogens. Gaseous chemicals, including acetic acid and natural plant compounds (Delaquis et al. 1999; Park et al. 2000; Weissinger et al. 2001), have also shown promise as treatments for reducing populations of pathogens on seeds intended for sprout production. To date, however, chemical treatments have not been described that eliminate all Salmonella and E. coli O157:H7 from inoculated alfalfa seeds.
The lack of success in killing pathogens by chemical treatment of alfalfa seeds has been attributed in part to the inability of aqueous solutions containing little or no surfactant to reach cells located between the cotyledon and testa, or in damaged areas of the seed. Scarification of seeds for the purpose of enhancing water uptake and rapid, uniform germination may confound this problem with some seed cultivars (Holliday et al. 2001). The hypothesis is that enmeshment of pathogens in scarified testae or damaged cotyledons may provide additional protection against contact with chemical antimicrobials.
The use of ultrasound to promote decontamination of raw vegetables has been described (Seymour et al. 2001). The cleaning action of cavitation appeared to remove Salmonella Typhimurium cells, rendering the pathogen more susceptible to chlorine. A study was undertaken with the objective of determining the combined effects of chemical, heat and ultrasound treatments in killing or removing Salmonella and E. coli O157:H7 on alfalfa seed with the hypothesis that combined stresses and enhanced exposure of cells to chemicals would result in higher lethality.
MATERIALS AND METHODS
Five serotypes of Salmonella enterica (Anatum, Cubana, Infantis, Montevideo and Stanley) were used. With the exception of Salm. Montevideo, which is an isolate from an infected patient in an outbreak associated with tomatoes, all strains were isolates from outbreaks of salmonellosis associated with alfalfa sprouts. Five E. coli O157:H7 strains were used: 932 (human isolate), 994 (salami isolate), E0018 (calf faecal isolate), H1730 (human isolate from an outbreak associated with lettuce) and F4546 (human isolate from an outbreak associated with alfalfa sprouts).
Preparation of inocula
Salmonellae and E. coli O157:H7 were grown in tryptic soy broth (TSB, pH 7·3; BD Diagnostic Systems, BBL/Difco) supplemented with 50 μg ml−1 nalidixic acid (TSBN; Sigma Chemical Co.). Cultures were incubated at 37°C and transferred (one loopful to 10 ml TSBN) three times at 24 h intervals immediately preceding use as inocula for alfalfa seeds. Populations of nalidixic acid-resistant Salmonella and E. coli O157:H7 in 24 h cultures were determined by serially diluting in sterile 0·1% peptone water, and surface plating duplicate 0·1 ml samples on tryptic soy agar (TSA; BBL/Difco) supplemented with nalidixic acid (50 μg ml−1; TSAN). Plates were incubated at 37°C for 24 h and colonies were counted.
Inoculation of alfalfa seeds
A 10 ml aliquot of 24 h TSBN cultures of each serotype of Salmonella was combined with 1 litre of deionized water (22 ± 1°C) and mixed for 30 s. Alfalfa seeds (1 kg, 22 ± 1°C) obtained from Caudill Seed Company, Louisville, Kentucky, USA, were added to the cell suspension and gently stirred for 1 min. The suspension containing the seeds was poured over a double layer of coarsely-woven cloth supported by a wire screen elevated approximately 5 cm above the work surface of a laminar flow hood. The seeds were spread in a layer approximately 0·5 cm thick, dried for 72 h, placed in a plastic bag and stored at 5°C for a minimum of 8 days before using in decontamination experiments. The same procedure was used for inoculating seeds with E. coli O157:H7.
Preparation of chemical treatment solutions
Five chemical treatments were evaluated for effectiveness in killing or removing Salmonella and E. coli O157:H7 on seeds: Ca(OH)2 (Sigma), 1% (w/v) in deionized water; Tween 80 (Sigma), 1% (v/v); 1% Ca(OH)2 plus Tween 80, 1%; Tsunami® 200 (Ecolab, Mendota Heights, MN, USA), 160 μg ml−1; and Fit® (Procter and Gamble Company, Cincinnati, OH, USA) powder, 0·5% (w/v). The peroxyacetic concentration in Tsunami solution was determined using a Tsunami test kit (Ecolab). Treatment of inoculated seeds in water served as a control.
Procedures for treating seeds
Water in an ultrasonic cleaner bath (Aquasonic Cleaner Bath, model 250D, VWR Scientific, West Chester, PA, USA) was adjusted to 23 or 55°C. Treatment solutions and water in the ultrasonic bath were degassed for 10 min immediately before seed decontamination experiments were carried out. The frequency of ultrasound fluctuated between 38·5 and 40·5 Hz during treatment of seeds.
The efficacy of treatment of seeds placed in plastic bags or in glass beakers was determined. A 20 ml volume of chemical solution or water (control) was placed in a Stomacher 80 bag (Seward Medical, London, UK) or a 600 ml glass beaker and placed in the ultrasonic bath at 23 or 55°C such that the level of the solution was below the level of the water in the bath. Inoculated seeds (5 g) were added to the chemical solutions or water and treated at 38·5–40·5 kHz for 2 or 5 min.
Immediately after treatment for 2 or 5 min, solutions were decanted from treated seeds, and 20 ml Dey-Engley (DE) neutralizing broth (BBL/Difco), followed by 20 ml 2×lactose broth (BBL/Difco) supplemented with 100 μg ml−1 nalidixic acid, were added to seeds inoculated with Salmonella; 20 ml DE broth and 20 ml 2× modified TSB (Padhye and Doyle 1991) (mTSB) supplemented with 100 μg ml−1 nalidixic acid were added to seeds inoculated with E. coli O157:H7. Approximately 100 seeds were removed from each bag or beaker to determine germination percentage using the procedure described by Weissinger and Beuchat (2000) before the seed and broth mixture was pummeled in a stomacher (Seward Medical) for 1 min. Undiluted stomachate (0·25 ml, in quadruplicate, and 0·1 ml, in duplicate) and stomachate (0·1 ml, in duplicate) serially diluted in sterile 0·1% peptone water were surface plated on TSAN. Plates were incubated at 37°C for 24 h and presumptive colonies were counted. Presumptive Salmonella colonies were randomly picked and tested for biochemical reactions using triple sugar iron (BBL/Difco) and lysine iron (BBL/Difco) agar slants. Presumptive colonies of E. coli O157:H7 were confirmed using the O157 latex agglutination test (Oxoid, Blasingstoke, UK) and API 20E diagnosis tests (BioMerieux, Hazelwood, MO, USA). The seed and broth mixtures were incubated at 37°C for 24 h. Cultures (1 ml) containing seeds inoculated with Salmonella were inoculated into 10 ml selenite cystine broth (BBL/Difco) and incubated at 37°C for 24 h. Selenite cystine broth cultures were streaked onto bismuth sulphite agar (BSA) (BBL/Difco) supplemented wit 50 μg ml−1 nalidixic acid, and plates were incubated at 37°C for 24 h before examining for presumptive Salmonella colonies. Confirmation of colonies was done as described above. Cultures of seed and mTSB mixtures incubated at 37°C for 24 h were streaked on sorbitol MacConkey agar (BBL/Difco) supplemented with 50 μg ml−1 nalidixic acid (SMAN). Presumptive E. coli O157:H7 colonies formed on plates incubated at 37°C for 24 h were confirmed as described above.
Removal of pathogens from seeds
The rate of release of Salmonella and E. coli O157:H7 from seeds as affected by ultrasound treatment (38·5–40·5 kHz) in deionized water at 23 and 55°C was determined. The procedure for treating seeds in bags or beakers was as described above. At 1 min intervals up to 10 min, samples of water were removed from the seed and water mixture and analysed for populations of Salmonella or E. coli O157:H7 using enumeration procedures described above. In addition, the rate of release of pathogens from seeds immersed in deionized water not subjected to ultrasound treatment was determined. The seed and water mixture was vigorously agitated in a water bath at 23 or 55°C during treatment. Samples of water were removed at 1 min intervals and analysed for populations of test pathogens.
Three replicate experiments for each set of experimental parameters were conducted. Data were subjected to the Statistical Analysis System (SAS Institute, Cary, NC, USA) for Analysis of Variance and Duncan's multiple range tests.
Seeds were treated in bags and beakers to determine whether container composition and geometry would influence the effectiveness of chemical and ultrasound treatment in killing Salmonella and E. coli O157:H7. Within each set of test parameters, the type of container did not influence the number of each pathogen surviving treatment.
With some exceptions, extending the ultrasound treatment at 55°C from 2 min to 5 min caused significantly (P≤ 0·05) higher lethality to Salmonella (Table 1) and E. coli O157:H7 (Table 2). The enhanced lethality of treatment time was less evident in solutions at 23°C compared with 55°C. In a few instances, higher numbers of pathogens were recovered from seeds treated for 5 min than from seeds treated for 2 min.
Combined effect of chemical, heat and ultrasound (38·5–40·5 kHz) treatments in killing Salmonella
on alfalfa seeds in bags and beakers
Combined effects of chemical, heat and ultrasound (38·5–40·5 kHz) treatments in killing Escherichia coli
O157:H7 on alfalfa seeds in bags and beakers
Ultrasound treatment of seeds in bags for 2 min at 23 or 55°C did not cause a reduction in seed germination percentage (Tables 1 and 2. Ultrasound treatment of seeds inoculated with Salmonella in beakers for 2 min, however, did result in a significant decrease in germination of seeds exposed to one of 12 chemical/temperature treatment combinations (Table 1), whereas treatment of seeds inoculated with E. coli O157:H7 in beakers for 2 min resulted in significant decreases in seed germination percentages of eight of 12 chemical/temperature treatment combinations (Table 2). The same lot of seeds was used to inoculate with Salmonella or E. coli O157:H7, so seed cultivar or other factors that may be attributed to different lots did not contribute to differences in retention of germination percentage. The initial moisture content of the two batches of inoculated seeds could have differed, however, to the extent that sensitivity to ultrasound may have been altered. The moisture content of inoculated seeds was not measured before combining with chemical solutions and subjecting to ultrasound treatment.
A comparison of data in Table 1 with results from studies using the same experimental protocol, with the exception that ultrasound was not applied during chemical treatment (Beuchat and Scouten 2001), indicates, overall, that ultrasound enhances removal and/or inactivation of Salmonella. Treatment of seeds at 23 or 55°C with 1% Ca(OH)2 was most effective in reducing populations without compromising seed viability. Treatment of seeds at 23°C with 160 μg ml−1 or 0·5% Fit also caused substantial reductions in populations of Salmonella without reducing seed germination percentage. Chemical treatments were generally less effective in killing E. coli O157:H7 on seeds (Table 2) compared with lethality against Salmonella (Table 1). Treatment of seeds inoculated with E. coli O157:H7 with 1% Ca(OH)2, with or without 1% Tween 80, resulted in the greatest reductions in populations of the pathogen without reducing seed germination percentage.
Experiments to determine the rate of release of Salmonella and E. coli O157:H7 in water, as affected by ultrasound treatment at 23 and 55°C, revealed that more cells were removed within the first minute of treatment when ultrasound was applied. Higher numbers were released from seeds in water at 23°C than in water at 55°C. This may be due, in part, to inactivation of the desiccation-stressed cells at 55°C. Ultrasound treatment did not have a marked effect on the release of cells of either pathogen when applied for an additional 9 min. In fact, populations decreased between 1 and 10 min of treatment of seeds in water held at 55°C.
Other researchers have also reported increases in populations of Salmonella and E. coli O157:H7 recovered from alfalfa seeds treated with chemical solutions for extended times (Jaquette et al. 1996; Beuchat et al. 2001). This phenomenon is attributed, in part, to greater infiltration of water into the seeds during extended exposure to chemical solutions, which may enhance the release of pathogens from seed components. Chemicals whose lethality is due to oxidizing activity are theoretically neutralized upon contact with seed tissue, thereby losing effectiveness in killing cells of pathogens lodged in subsurface areas. Water that permeates seeds would enhance detachment and removal of cells upon subsequent stomaching in neutralizing solution or diluent. Thus, the increase in number of viable cells surviving on seeds subjected to extended treatment time is only apparent. This aberration is generally less evident when seeds are treated with higher concentrations of chemicals.
Treatment of alfalfa seeds at 55°C was more lethal to pathogens than treatment at 23°C, regardless of the chemical sanitizer. However, treatment at 55°C for 5 min also tended to reduce the germination percentage of seeds. Jaquette et al. (1996) reported that heating alfalfa seeds at 54°C for 5 min did not significantly reduce Salmonella Stanley populations. Treatment for 10 min at 54°C reduced the viable cell population by 98%. With the exception of Fit, Beuchat and Scouten (2001) evaluated the same chemicals used in the present study for their effectiveness in killing the same Salmonella serotypes at 23 and 55°C on seeds not treated with ultrasound. Their study revealed that treatment at 55°C for 10 min caused a reduction in seed germination percentage. With the exception of treatment of seeds with 1% Ca(OH)2 plus 1% Tween 80, treatment at 55°C for 5 min did not significantly reduce germination. These observations suggest that heat, in combination with ultrasound, is more detrimental to seed viability compared with heat treatment without ultrasound.
The overall results of this study indicate that ultrasound treatment of alfalfa seeds at 38·5–40·5 kHz enhances the effectiveness of chemical sanitizers in killing Salmonella and E. coli O157:H7. Heat also contributes to an increase in the removal of, or lethality to Salmonella and E. coli O157:H7 on alfalfa seeds. However, the combined effects of ultrasound and heat in reducing populations of pathogens are not dramatic. With the exception of, perhaps, irradiation, a set of treatment conditions that will kill consistently more than ≈ 4 log10 cfu g−1 of Salmonella or E. coli O157:H7 on alfalfa seeds without reducing germination percentage has not been described.