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It’s pretty well established that cruciferous vegetables like broccoli have cancer-preventative compounds. Broccoli sprouts specifically are a source of glucoraphanin, which creates sulforaphane when chewed or swallowed. That compound accelerates the body’s ability to detoxify from various pollutants.

The researchers had about 300 Chinese men and women living a rural community in Jiangsu Province, China, drink a beverage of sterilized water, pineapple, lime juice and dissolved freeze-dried broccoli sprout powder. The control group drank a mixture without the sprout mixture. All the participants had their urine and blood tested, and when they did, the scientists discovered that among the participants drinking the broccoli beverage, the rate of excretion of the carcinogen benzene increased 61% and the rate of excretion of the irritant acrolein rapidly increased 23%.

“The situation is that people throughout China are breathing dirty air, and the exposure is largely unavoidable,” says study author Tom Kensler. “We wanted to boost the defense mechanism that accelerates the rate that these are cleared form the body so there is less opportunity for harm to be evoked by chemicals.”

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Plants for seed production are grown in typical agricultural environments and seed are generally treated as a raw agricultural product. Potential sources of contamination in the field include agricultural water, improperly managed animal manure, contact with wild animals, and inadequate worker hygiene. In addition, domestic animals may be allowed to graze on alfalfa fields. While such contact is not likely to be a significant problem for the primary use of seed, i.e., seed for forage production, even low level, sporadic contamination of seed for food use may result in significant public health concerns because the sprouting process amplifies pathogen levels.

The CDHS/FDA survey found sprouts being produced in buildings, sheds, greenhouses, modified buses, agricultural fields, or a combination of these. The 45 firms covered by the inspection survey produced 24 different types of sprouts, the most frequently observed products were mung bean, alfalfa, clover, and radish sprouts. Only 25 firms reported producing over 5,000 pounds of sprouts per day (CDHS/FDA, 1998).

Sprout production consisted broadly of the steps depicted in Figure 2. Some firms may add to or omit some of these practices depending on a number of factors, including the type of seed being sprouted and the size and resources of the firm. Sprout production is described in more detail in Appendix 2.

Although seed appears to be the primary source of contamination in sprout-associated foodborne illness outbreaks, practices at the sprouting establishment may increase or decrease the extent of the microbial hazards. Poor sanitation and inadequate hygiene at the sprout production facility can exacerbate the problem of sprout-associated foodborne illness. The CDHS/FDA survey indicated there were significant gaps in sprout manufacturers' understanding and knowledge of food safety, GMPs, and regulatory requirements. In addition, most sprout producers were not registered as food processing establishments as required by California regulations, and thus had not been previously inspected for compliance with GMPs.

Examples of gaps in knowledge of GMPs observed during the inspection survey include, but are not limited to:

Raw materials and other ingredients should be inspected upon receipt to ensure they are clean and suitable for processing into food. Bags of seed which have been contaminated with rodent urine will glow when viewed using a blacklight. This is a useful tool to ensure incoming product is clean and to monitor contamination by rodents during seed storage. However, at the time of the survey, the majority of firms did not blacklight or visually inspect incoming seed.
Seed should be stored under conditions that will protect against contamination and prevent deterioration.
At the time of the survey, less than half of the firms applied disinfection treatments to seed before sprouting.
Approximately 22% of firms used non-municipal well water. Half of these firms did not test water for microbial quality.
Many firms did not have hot water for cleaning equipment or for hand washing.
At the time of the survey, many firms were observed to have equipment that was not easily cleaned and/or was not properly stored.
Five firms had no cooling facilities for holding finished product. Another five firms had coolers above 45°F.
Approximately half of the firms surveyed reported that employees had not received hygiene or sanitation training in the last year. Almost as many firms reported that they did not currently supervise employee hygiene or sanitation. Inspectors noted that basic sanitary knowledge was lacking for many food workers who were observed handling unsanitary objects and then handling sprouts.
Restrooms at some facilities were inaccessible, inadequately stocked or maintained. Few sprout production facilities had hand dip stations.
None of the firms surveyed had sufficient records to facilitate a complete traceback from finished product to the field where the seed was grown.

Sources of seed contamination in the agricultural environment are similar to those described for fresh produce (NACMCF, 1998). The likelihood of contamination in the field may be reduced by systematic implementation of GAPs.

The FPWG found little scientific information available on the impact that seed conditioning and handling practices have on the microbiological safety of sprouted seeds. However, as noted earlier, opportunities for contamination or cross contamination exist. Furthermore, damage to seed may make subsequent removal of pathogens more difficult. Risk of contamination of seeds with foodborne pathogens at this stage might be reduced by development of systematic procedures to minimize contamination or cross contamination.

Seed storage

As noted earlier, once pathogens are present in seeds, they are likely to survive for extended periods of time under normal seed storage conditions. Several researchers have investigated pathogen survivability on seed stored under different conditions (Jaquette et al., 1996, Taormina and Beuchat, 1999). In one study, S. Stanley was inoculated onto alfalfa seeds at 102-3 CFU/g. Salmonella populations decreased 1 log after 8 - 9 weeks storage at 8°C. Increasing storage temperature to 21°C resulted in a 2 log reduction in S. Stanley after 9 weeks (Jaquette et al., 1996). In another study, populations of E. coli O157:H7 inoculated onto alfalfa seeds (initial level of 103 log CFU/g) remained relatively unchanged after 38 weeks storage at 5°. However, within one week of storage at 25 and 37C, E. coli O157:H7 populations decreased significantly. When seeds were stored at 5, 25, or 37°C for 54, 13, or 8 weeks respectively, pathogens were not detectable by direct plating; however, culture enrichment revealed the presence of pathogens in seeds after 38 weeks of storage at 25 and 37°C but not after 54 weeks (Taormina and Beuchat, 1999).

Seed testing

Although epidemiological investigations have frequently identified seeds as the most likely source of contamination of sprouts, laboratory analyses have often been unable to isolate pathogens from implicated seed. This suggests that contamination may be sporadic and at low levels. In the 1995-1996 S. Newport outbreak, analysis of the implicated seed lot by MPN yielded 0.1-0.6 CFU of S. Newport/25 g of seed (4 - 24 cells/1000 g seed) (Van Benden et al., 1999). Analysis of the seed implicated in the 1998 S. Havana outbreak revealed S. Tennessee, S. Cubana, and S. Havana at levels of approximately 4 CFU/1,000 g (Farrar and Mohle-Boetani, 1999). Nonetheless, sprout producers may find advantages to testing seeds for pathogens. While a negative result does not guarantee the absence of pathogens, a positive result would allow a producer to avoid using seed lots that have been shown to contain pathogens.

Seed decontamination

In general, sanitizing is more effective for reducing contamination on seeds than on sprouted seeds (Caetano-Anolles et al., 1990). This may be due to a combination of lower levels of both microorganisms and organic material present on seeds than on sprouts, and the internalization of bacteria into sprout tissues during sprouting making them physically inaccessible to sanitizers (Hara-Kudo et al., 1997; Itoh et al., 1998).

A successful seed decontamination treatment must inactivate microbial pathogens while preserving seed viability, germination, and vigor. Seeds vary in sensitivity to antimicrobial agents and other treatments, which determines how well they germinate and grow after treatment. In addition, a treatment that is effective for one type of seed may not be applicable to all types of seeds. Seeds vary in surface features, which may influence how well an antimicrobial agent can access and inactivate pathogens on or in the seed. Washing seed with water alone decreases levels of B. cereus, E. coli, or Salmonella by 1 log and hence is only marginally effective in reducing pathogens (Harmon et al., 1987; Potter and Ehrenfeld, 1998). Several investigators indicated to the FPWG that alfalfa seeds are generally the most difficult type of seed to sanitize effectively.

A number of researchers have investigated the ability of chlorine compounds to inactivate pathogenic bacteria on seeds, particularly alfalfa seeds. Pretreatment of the seeds prior to germination with 150 ppm of hypochlorite did not eliminate S. Newport (Aabo and Baggesen, 1997). Jaquette et al. (1996) examined the effects of chlorine (sodium hypochlorite treatments ranging from 100 - 2040 ppm active chlorine) on alfalfa seeds inoculated with 102-3 CFU of S. Stanley/g. Treating seed with 100 ppm active chlorine solution for 5 or 10 minutes significantly reduced S. Stanley. A further reduction, but not elimination, occurred after treatment with 290 ppm active chlorine. Treatment with 1,010 ppm active chlorine did not result in additional reduction compared to 290 ppm. Treatment with 2,040 ppm active chlorine reduced S. Stanley to undetectable levels (<1 CFU/g). A study of disinfection procedures for rice seeds showed that ethanol, hydrogen peroxide, or 1,000 ppm sodium hypochlorite reduced APCs by 2-3 logs, but did not eliminate microflora (Piernas and Guiraud, 1997).

In another study, alfalfa seeds were inoculated with a mixture of 5 Salmonella serovars and subjected to a variety of treatments at different concentrations using a variety of antimicrobial chemicals (i.e., calcium and sodium hypochlorite, hydrogen peroxide, and ethanol) (Beuchat, 1997). Significant reductions in Salmonella populations were observed with most increases in concentration of the test chemical. No adverse effects on percent germination were observed for any treatment at any concentration. Treatment solutions containing calcium hypochlorite and sodium hypochlorite at concentrations of 1,800 and 2,000 ppm active chlorine, respectively, 6% hydrogen peroxide, or 80% ethanol were effective in reducing populations by 1000-fold after a 10 minute treatment (Beuchat, 1997). Consistent with Jaquette et al. (1996), Salmonella were not detected by direct plating after treatment with 2,000 ppm active chlorine. However, in this study, where seed disinfection treatments resulted in < 1 CFU Salmonella/g, enrichment procedures were employed, and, in all cases, viable Salmonella were recovered. The author speculated that Salmonella trapped in cracks and crevices on the seed were inaccessible to lethal concentrations of chemicals (Beuchat, 1997).

In a subsequent study, alfalfa seeds inoculated with E. coli O157:H7 at 1-2 X 106 were treated for 10 minutes with 2.0%, 2.5%, or 3.0% (w/v) calcium hypochlorite solutions containing 13,800, 17,000, or 20,670 ppm active chlorine, respectively (Fett, 1998). The lowest concentration (2.0%) resulted in a 2 log reduction while higher concentrations (2.5 and 3.0%) reduced E. coli O157:H7 by approximately 4 logs. No viable bacterial cells were detected on seed treated with 2.5 or 3.0% calcium hypochlorite when plating was done on selective E. coli/coliform Petrifilm count plates. However, survivors were recovered by both direct plating onto Tryptic Soy Agar (TSA) as well as by enrichment. Plating on both selective and nonselective medium indicated that some of the viable cells of E. coli present after treatment with calcium hypochlorite were injured. The author suggested that additional reductions may occur when chemical treatment is used in combination with physical treatments such as low dose irradiation or heat (Fett, 1998).

Another researcher treated seed with sodium- or calcium hypochlorite, ozonated water and hydrogen peroxide. Of these treatments, 2% calcium hypochlorite showed the greatest reduction but did not completely eliminate the natural microflora. Sodium hypochlorite at concentrations greater than 1% inhibited seed germination. Treatment of over a dozen types of seeds in 2% calcium hypochlorite or 6% hydrogen peroxide resulted in variable germination rates. For example, 2% calcium hypochlorite allowed good germination of alfalfa seeds but drastically reduced germination of onion seeds (Moline, 1999).

Alfalfa seeds were artificially contaminated with E. coli O157:H7 and the effectiveness of sodium hypochlorite, calcium hypochlorite, hydrogen peroxide, ethanol, chlorine dioxide, acidified sodium chlorite, and a consumer product (Vegi-CleanTM, Microcide, Inc., Detroit, Mich.) were tested for produce sanitation. In no instance was E. coli O157:H7 eliminated (L. Beuchat, personal communication). Calcium and sodium forms of hypochlorite at a concentration of 2% appear to be equally effective for E. coli O157:H7 inactivation. However, calcium hypochlorite may be preferable to sodium hypochlorite because it is not as volatile. In addition, calcium hypochlorite solutions up to 20,000 ppm did not affect percent germination of alfalfa seeds. However, at high concentrations, alfalfa seed vigor (rate of germination) was slowed (Beuchat, 1999). Percent germination of alfalfa seed treated for 10 minutes in 3 % calcium hypochlorite solution was not significantly different from untreated seed after 2 days of sprouting, however, the rate of germination was slower (Fett, 1998).

The effectiveness of few other sanitizing agents for reducing the pathogen levels on seeds has been reported in the literature. A 1 - 2 log reduction in microbial counts on rice seed was achieved with a treatment with 1% hydrogen peroxide (Piernas and Guiraud, 1997b). Treatment with 70% ethanol was effective for reducing pathogen levels but significantly inhibited germination. Reducing the ethanol concentration to 10% improved germination but decreased its microbiocidal effect (Piernas and Guiraud, 1997b). Percent germination of alfalfa seed was also significantly decreased by treatment with 30 or 70% ethanol, precluding the use of ethanol as a sanitizer (Taormina and Beuchat, 1999).

Preliminary trials with commercially available sanitizers, such as GlycorineTM (a mixture of lauric acid and sodium lauryl sulfate), have been found to be effective in reducing E. coli O157:H7 by > 2 logs per gram of alfalfa seeds. Trials with these sanitizers have been at least as effective as limited trials with hydrogen peroxide or other chemical seed treatments. Research is continuing to optimize exposure time and concentrations (Slade, 1999). TsunamiTM (active oxygen solution, Ecolab, Mendota Heights, Minn.) and VortexTM (active oxygen solution, Ecolab) at 80 ppm and Vegi-CleanTM at 20,000 ppm for 10 minutes reduced E. coli O157:H7 to < 0.5 CFU/g on alfalfa seeds. However, E. coli O157:H7 was detected from treated seeds after culture enrichment (Beuchat, 1999).

Ozone and ozonated water decreased microbial levels without adversely affecting the sprouting of black matpe and alfalfa seeds; and decreased the natural microflora on beans, peas, grain and spices by 1 - 3 logs (Naito and Shiga, 1989; Naito et al., 1988). Hydronium ion treatment of alfalfa seed has been initiated (Slade, 1999).

Potassium sorbate, calcium propionate, gallic acid, benzoic acid, salicylic acid, dihydroxybenzoic acids, hydroxybenzoic acids, riboflavin, phloxine b, eosin b, nisin, and calcium-EDTA were also tested. Of these treatment options, only calcium-EDTA and 4-hydroxybenzoic acid appear to warrant further study (Moline, 1999).

Surfactants

It has been suggested that the barrier to disinfecting seeds is not in the lethality of the treatment solutions but in the ability of treatments to reach pathogens in the seeds. Microbial cells in seed crevices may be protected from exposure to lethal concentrations of sanitizers and related surface treatments (Caetano-Anolles, 1990; Beuchat, 1997). The use of surfactants alone reduced E. coli O157:H7 populations by approximately 1 log (Beuchat, 1999). Pre-treatments or co-treatment with surfactants have been proposed as a possible means of improving the effectiveness of sanitizing agents. Early research has not been promising. The use of surfactants Tween 80 and benzalkonium chloride had little effect on improving decontamination of rice seed (Piernas and Guiraud, 1997b) or alfalfa seed (Beuchat, 1999) compared to disinfectant treatments alone. However, research is continuing in this area..

Heat

Heat treatments have also been explored as a possible means for reducing pathogen levels on seeds. S. Stanley levels were unchanged after a 10 minute soak in water at 21°C. Soaking seed in 54°C water for 5 or 10 minutes significantly reduced but did not eliminate the pathogen. Soaking seed at 57 or 60° 5 minutes reduced S. Stanley to < 1 CFU/g, without substantial loss of germination. Higher temperatures and longer times (> 5 minutes) caused significant declines in germination. Treatments up to 66°C for 10 minutes caused germination to decline to 6% (Jaquette et al., 1996). The narrow temperature range between treatment efficacy and seed injury may make relying on heat alone to eliminate pathogens difficult on a commercial scale.

Heat and disinfectants

It has been suggested that pathogens that survive chemical disinfection treatments may be injured (Fett, 1998). Microbial populations (APCs) on rice seed decreased at least 5 log cycles when a 5-minute sodium hypochlorite soak was combined with heating at 60°C for 5 minutes (Piernas and Guiraud, 1997b). Alfalfa seed were inoculated with E. coli O157:H7 and treated with various solutions at 55°C for 3 minutes. Treatment with calcium hypochlorite (20,000 ppm active chlorine) eliminated pathogens from three of three samples as determined by direct plating. When seeds were subjected to two 3-minute rinses at 55°C and at the same concentration of calcium hypochlorite, E. coli O157:H7 was detected in one of three samples. The pathogen was detected in all samples after culture enrichment (Taormina and Beuchat, 1999).

In most patents associated with sprouting systems, the decontamination step, if listed, is recommended primarily to inhibit spoilage rather than for pathogen reduction. One U.S. patent, issued for sterilizing and cultivating seeds for sprouting, describes a procedure in which the seeds are exposed to heat for a short period, quickly cooled, and then soaked in chlorine solution (Suzuki and Takizawa, 1997). This treatment reduced but did not eliminate pathogens. Precise treatment conditions were not provided because of variability in the effects of heat on viability of different types of seeds, but 70°C for 20-30 seconds or 90°C for 10 seconds was provided as a guideline.

Radiation

Gamma irradiation has been shown in preliminary studies to be an effective antimicrobial treatment for both seeds and sprouts. Using Cs137 and temperature control (5°C), E. coli O157:H7 and Salmonella were inactivated while seed viability was maintained. However, the dose required to inactivate these pathogens (up to 5 kGy) exceeds current allowable limits (1 kGy) for treatment of produce. Use of gamma irradiation in conjunction with chemical treatments (e.g., 20,000 ppm calcium hypochlorite) is being investigated (Thayer, 1999).

Preliminary trials with electron beam irradiation of alfalfa seeds at 2.5 - 10 kGy at various accelerated voltages appear promising. Researchers observed a linear reduction in the levels of Salmonella and natural microflora at increasing dose levels. Pathogens were not detected in seeds treated at 10 kGy. However, enrichment was not done prior to analysis. Percent germination was not reduced at the highest radiation level tested. However, researchers noted physiological changes (i.e., shortening, thickening, and curling) in the roots of sprouts grown from treated seeds. Additional research on depth of penetration and combining electron beam irradiation at different levels with chemical treatments is planned (Slade, 1999).

Other

The use of a number of physical, non-thermal processing technologies, alone or in combination with antimicrobial chemicals, is virtually unexplored for seed decontamination. Several relatively inexpensive technologies have not been investigated but may have potential use in sprout production. Ultraviolet light might be useful for surface disinfection of seeds, and its activity may be enhanced by antimicrobial chemicals such as hydrogen peroxide. At certain ultrasound frequencies, light is produced (soniluminescence), which might be useful in peroxide activation for microbial control. Flashes of bright white light (PureBright® technology) have been effective for killing microorganisms on food surfaces. The feasibility of using high pressure processing for reducing microbial levels on vegetables has been shown (Arroyo et al., 1997). Pulsed electrical or magnetic field technologies are additional processes that have not been tested and might be applicable to seeds. Inactivation of microbial cells lodged in seed crevices might be enhanced by treatment of the seeds with gaseous antimicrobial compounds (e.g., gas phase peroxide). Incorporation of these compounds into packaging materials might also be useful for controlling microbial growth after sprouting. In addition, vacuum infiltration of calcium or sodium hypochlorite may allow sanitation of surfaces of seeds that have been resistant to current treatments.

Seed disinfection treatments may be done by the seed supplier or by the sprout producer. Some types of treatments may be more easily done at one location than another. In addition, because of the potential for survivors or for recontamination of treated seed, sprout producers may need to implement additional treatments just prior to sprouting.

A reduction in pathogens of 1 - 2 logs has little practical significance because pathogens surviving on seeds would be expected to grow during the spouting process. Some disinfection processes (e.g., 20,000 ppm calcium hypochlorite) have been identified that achieve higher reductions. However, to date, no single treatment has been identified that can eliminate pathogens, if present, or one that can be applied to all types of seed. It is possible that combining treatments may provide sufficient cumulative reductions to meet food safety goals.

Treatments/interventions during the sprouting process

It may be possible to implement treatments during the sprouting process to prevent or inhibit the growth of pathogens that survive seed treatments. However, there is little published literature in this area. Use of rinse water, chlorinated at 100 ppm, during sprouting of mung beans decreased counts of the natural microflora by < 1 log (Splittstoesser et al., 1983). Preliminary trials have begun for a number of other disinfectant treatments that might be applied to sprouts or irrigation water during sprouting. Treatments under investigation include the use of sodium and calcium hypochlorite, hydrogen peroxide, TsunamiTM, sodium EDTA, and sodium chlorite (Fett,1999). As with seed, successful treatments will need to be effective in killing pathogens without being phytotoxic to growing sprouts.

Biofilms tend to protect entrapped foodborne pathogens from the antimicrobial activity of sanitizers and may also interfere with the activity of disinfectants applied to sprouted seed during or after sprouting. Studies on biofilm formation on sprouts have been initiated. Scanning electron microscopy has shown extensive formation of biofilms on commercially obtained sprouts and on 4-day old sprouts grown in the laboratory (Fett, 1999).

Competitive exclusion techniques, where non-pathogenic microorganisms are used to repress the growth of pathogenic bacteria during sprouting have been suggested; however, there are few reports in the literature. When co-inoculated with L. monocytogenes, nisin-producing lactococci isolated from bean sprouts reduced the levels of the pathogen by 1 log (Cai et al., 1997). Work has been initiated on using competitive exclusion during sprouting (Fett, 1999). Naturally occurring microbes from commercial alfalfa sprouts were isolated and tested for their ability to repress the growth of a mixture of Salmonella strains. Preliminary trials have shown 1 - 3 log reductions in Salmonella counts during a 7-day grow-out after challenge with the various microflora isolates.

While contamination of seeds may be at low levels and difficult to detect, microbial populations increase significantly during the sprouting process (Jaquette et al., 1996). Significant levels of microorganisms are recovered in the irrigation water during sprouting. Testing of sprout irrigation water for total counts and coliforms indicate that the water contains at least 90 % of the counts found in the sprouts themselves. It is possible that testing spent irrigation water may be useful for monitoring microbial levels and detecting pathogens that may be present during sprouting. Research is needed to determine an optimum testing protocol, including when and how to test, to maximize accuracy and achieve economic feasibility. The effect of disinfectant treatments applied during sprouting on the level of pathogens and microflora in spent irrigation water is unknown. If disinfectants kill pathogens in irrigation water but allow pathogens to survive in sprouts, testing of irrigation water would be meaningless (Tortorello and Fu, 1999).

Interventions for sprouted seed

There have been few studies on reducing pathogen levels in finished product. Washing mature sprouts with water decreased levels of B. cereus, E. coli or Salmonella by no more than 1 log (Harmon et al., 1987; Potter and Ehrenfeld, 1998). Soaking of fully developed mung bean sprouts for 30 minutes in 0.5% sodium hypochlorite resulted in a 2 log decrease in counts (Splittstoesser et al., 1983). Treating mung bean sprouts with 500 ppm sulfur dioxide reduced microbial counts by 1 - 2 log (Splittstoesser et al., 1983).

As noted earlier, gamma irradiation has been shown in preliminary studies to be an effective antimicrobial treatment for both seeds and sprouts. However, the dose required to inactivate these pathogens (up to 5 kGy) exceeds current allowable limits (1 kGy) for treatment of produce (Thayer, 1999).

As indicated above, sprouted seeds represent a unique microbial food safety concern due to the potential for certain pathogenic bacteria to grow rapidly during the germination and sprouting of the seeds. For sprouts contaminated by pathogens that do not grow during sprouting (protozoa, viruses), the risks of adverse public health consequences are similar to those already noted for fresh produce (NACMCF, 1998). In fact, the risks associated with those microorganisms might be reduced due to the extensive washing that sprouts receive during their production. Thus, pathogenic bacteria that either cannot grow under the conditions encountered during sprout production (e.g., Campylobacter jejuni) or that are not likely to be competitive enough to reach the levels needed to have an adverse public health impact (e.g., Staphylococcus aureus) are not considered to be an increased risk in sprouts compared to other fresh produce.

The following are pathogens that have either been implicated with sprouted seed-associated outbreaks or have been identified as being a potential source of increased risk due to their ability to proliferate during sprouting.

Salmonella

As noted in the preceding section, Salmonella has been responsible for several large sprout-associated outbreaks worldwide (O'Mahony et al., 1990; Oregon Health Division, 1995; Mahon et al., 1997; Puohiniemi et al., 1997; Farrar and Mohle-Boetani, 1999). Reported outbreaks of salmonellosis associated with sprouted seeds suggest an initial low level contamination of the seeds, followed by growth during sprouting (Splittstoesser et al., 1983; Mahon et al., 1997). Salmonellae have been shown to grow during the sprouting process (Splittstoesser et al., 1983; Andrews et al., 1983).

Enterohemorrhagic Escherichia coli

Enterohemorrhagic E. coli have been responsible for several large sprout-associated (radish and alfalfa) outbreaks (CDC, 1997; Wantabe and Okasa, 1997). Epidemiological and/or microbiological evidence suggest that seeds were the source of the pathogen (Itoh et al., 1998). E. coli O157 can grow rapidly to large populations during sprout production.

Listeria monocytogenes

L. monocytogenes is associated with soil, plant and animal products, and food processing environments. Because it is so ubiquitous, there are multiple opportunities for L. monocytogenes to contaminate either seeds or sprouts. L. monocytogenes can grow at refrigeration temperatures on a variety of produce, including sprouts (Buchanan, 1999; Lovett, 1989). This pathogen has been isolated from commercially produced sprouted seeds but no cases of human listeriosis have been linked to those sprouts. Whether the growth amplification kinetics seen with Salmonella and E. coli O157 during the sprouting process also occur with L. monocytogenes is unknown. At the present time, it is unclear whether the presence and growth of L. monocytogenes in sprouts is significantly different from the presence and growth of L. monocytogenes in other fresh produce.

Bacillus cereus

B. cereus is a ubiquitous spore-forming bacterium. It is commonly found in soil and on plants (Kramer and Gilbert, 1989). In 1973, an outbreak was associated with the consumption of sprouts (a mixture of soy, cress, and mustard seeds packaged in a seed sprouting kit) contaminated with B. cereus. As with other pathogens associated with outbreaks from contaminated sprouts, the likely source was seed. B. cereus is capable of growth under seed sprouting conditions (Harmon et al., 1987).

Yersinia enterocolitica

Yersinia enterocolitica can be found in diverse foods of animal origin including pork, beef, poultry, and dairy products, and is commonly isolated from different environments such as lakes, rivers, wells, and soil (Kapperud, 1991). An outbreak of yersiniosis was associated with eating non-commercially produced bean sprouts that were grown using pond water (Cover and Aber, 1989). As a psychrotroph, it must be assumed that Y. enterocolitica can grow on sprouted seeds during refrigerated storage (Chao et al., 1988) just as it can with other products. The bacterium is likely to be capable of growth during sprout production, but no specific data are available.

Shigella

Shigella, because of its low infectious dose (10-100 organisms), its dissemination in fecally-contaminated water, its ability to proliferate in vegetables, and the high degree of handling often associated with the production and packaging of sprouts, must be assumed to be a potential cause of foodborne disease in sprouted seeds (Rafii et al., 1995). However, no reports of this pathogen linked to foodborne disease outbreaks associated with sprouted seeds were identified.