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Applied and Environmental Microbiology, June 2002, p. 3114-3120, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.3114-3120.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

A. O. Charkowski,1* J. D. Barak,2 C. Z. Sarreal,2 and R. E. Mandrell2

Department of Plant Pathology, University of Wisconsin—Madison, Madison, Wisconsin 53706,1 Produce Safety and Microbiology Research Unit, USDA Agricultural Research Service, Albany, California 947102

Received 26 September 2001/ Accepted 28 February 2002


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ABSTRACT
 
Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log10 on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log10. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.


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INTRODUCTION
 
Sprout producers in North America, Asia, and Europe have faced numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks since 1995 (2, 17). Several of the outbreaks were traced to sprouts grown from seeds contaminated with low levels of human pathogens (2, 15, 19, 20, 27).

As a result of the many recent outbreaks traced to sprouts and guidance by a U.S. Food and Drug Administration document (2), many producers now sanitize their sprout seeds with 20,000 ppm of calcium hypochlorite before sprouting and test each crop of sprouts for S. enterica and E. coli O157:H7. Epidemiological evidence suggests that this sanitation protocol may prevent some, but not all, outbreaks (2, 5), and experimental sanitation of naturally contaminated alfalfa seeds did not eliminate S. enterica from the seeds (23). To date, no seed sanitation method has been shown to eliminate S. enterica or E. coli O157:H7 from laboratory-contaminated seeds (4, 13, 14, 19, 24, 25). In addition, the recommended calcium hypochlorite method does not kill or remove all naturally occurring nonpathogenic bacteria from alfalfa seeds, suggesting that bacteria on seeds may be in locations inaccessible to calcium hypochlorite treatment (7).

Sprouts present an unusual food safety predicament compared to other fresh produce because bacteria, including S. enterica and E. coli O157:H7, may multiply by several logs on the sprouting seeds during the first few days of germination (1, 22, 23). Experiments with radish and alfalfa sprouts grown from laboratory-contaminated seeds have demonstrated that human pathogens may be present between plant cells inside sprouts, where they can resist decontamination treatments (9, 12). Washing sprouts in water only slightly reduces the number of bacteria found on the sprouts, and green sprouts are usually not cooked before being eaten. Thus, sprouts are a good vehicle for food-borne pathogens (1).

Although other workers have reported that both S. enterica and E. coli O157:H7 grow on alfalfa sprouts (1, 22, 23), there have not been comparative studies examining differences in how these two species grow in association with alfalfa sprouts. We examined the effects of temperature, initial inoculum dose, and frequency of irrigation on S. enterica and E. coli O157:H7 levels on sprouts. We found that S. enterica grows to higher levels on alfalfa sprouts than E. coli O157:H7. We also found that frequently changing the irrigation water reduces the number of E. coli O157:H7 cells but not the number of S. enterica cells associated with the alfalfa sprouts, suggesting that there are significant differences in how these two pathogens grow in association with sprouting seeds. We transformed S. enterica and E. coli O157:H7 strains isolated from contaminated seed lots or other environmental sources with a plasmid that expresses the green fluorescent protein (GFP). Since bacteria expressing the GFP do not require additional cofactors to fluoresce (6), we were able to view bacteria easily on intact sprouts with a fluorescence stereomicroscope and a confocal laser scanning microscope (CLSM). We found that the S. enterica and E. coli cells were attached mainly to alfalfa sprout roots, that only S. enterica was attached to sprouts in large cell aggregates, and that only S. enterica was attached in large numbers to sprout seed coats.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth media.
Plasmid pKT-kan is a broad-host-range stable vector in which a 131-bp nptII promoter fragment from Tn5 is fused to the GFP gene of pPROBE-KT (16). S. enterica and E. coli O157:H7 strains (Table 1) were transformed by electroporation with pKT-kan. S. enterica serovar Newport strain 96E01153C-TX and E. coli F4546 transformed with pKT-kan were used for the majority of the experiments, and these strains were designated SeN(pKT) and Ec(pKT), respectively. Bacteria were grown on Luria-Bertani (LB) medium plus kanamycin (40 µg/ml), Salmonella-Shigella (SS) medium, or sorbitol MacConkey (SMAC) medium. All media were obtained from Difco Laboratories (Detroit, Mich.).


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  		TABLE 1. Bacterial strains used in this study

Alfalfa seeds.
Seeds for sprouting were obtained from Geo. W. Park Seed Co., Inc. (Greenwood, S.C.), Johnny's Selected Seeds (Albion, Maine), and International Specialty Supply (Cookeville, Tenn.).

Inoculation and sprouting of alfalfa seeds.
Before inoculation, alfalfa seeds were treated with 3% (wt/vol) calcium hypochlorite (Fisher Scientific, Springfield, N.J.) at a ratio of 1 g of seeds to 5 ml of calcium hypochlorite for 15 min and then rinsed at least three times with sterile water. Approximately 50 seeds were placed in a petri plate with 20 ml of water, and the seeds were incubated at room temperature on a rotating shaker at 40 rpm. The water in which the germinating seeds were soaked is referred to below as the irrigation water. For growth curve and microscopy experiments, all of the irrigation water was removed from the petri plates with a pipette after 1 h and replaced with 20 ml of a suspension containing 106 CFU of bacteria per ml in water. We did not extensively wash the sprouting seeds since we were attempting to model commercial sprout production, in which the sprouts are constantly tumbling through a pool of water that is slowly dripping from spray nozzles in a sprouting drum. The inoculum was removed from the petri plates with a pipette and replaced with 20 ml of sterile water after approximately 3 to 4 h of incubation with the alfalfa seeds. Seeds were incubated at room temperature on a rotating shaker for 2 to 3 days, and the irrigation water was removed and replaced daily with 20 ml of sterile water. Sprout samples were taken daily, and the number of CFU per sprout was determined as described below.

To determine the effects of inoculum dose and incubation temperature on S. enterica and E. coli O157:H7 levels on sprouts and in sprout irrigation water, alfalfa seeds were sanitized with calcium hypochlorite as described above. The seeds were inoculated with 20 ml of SeN(pKT) or Ec(pKT) bacterial suspensions containing 1, 10, 102, 103, or 104 CFU/ml of water. The petri plates containing the germinating seeds were placed on a rotating shaker at 40 rpm and incubated for 2 days at 20, 30, or 35°C. The irrigation water was not replaced with fresh water during the course of these experiments. Water and sprout samples were taken 2 days after the seeds were inoculated, and the number of CFU per milliliter of water and the number of CFU per sprout were determined as described below.

For trials examining the effects of frequent replacement of irrigation water, approximately 50 alfalfa seeds were prepared as described above and inoculated with 20 ml of a 103-CFU/ml SeN(pKT) or Ec(pKT) bacterial suspension, and the seeds were germinated at 20°C. The irrigation water was changed approximately every 10 h. During each water change, the sprout irrigation water was removed from the plates and discarded, the sprouting seeds were rinsed once with approximately 20 ml of sterile water, and then 20 ml of sterile water was added to the sprouting seeds in the petri plates. The sprout irrigation water was changed as described above 2 h before sprout and water samples were analyzed for bacterial levels.

Determination of bacterial levels on sprouts and in irrigation water.
Three to five samples were examined per time point for each experiment, and all experiments were repeated at least three times. To determine the number of CFU per sprout, the sprouts were rinsed once with water, and then individual sprouts were added to 500 µl of 1x phosphate-buffered saline (PBS) (pH 7.4) and homogenized with a pestle connected either to a cordless electric drill (Black and Decker, Hampstead, Md.) or a MINIMITE cordless tool (Dremel, Racine, Wis.). Tenfold dilutions of the homogenate were prepared in 1x PBS (pH 7.4) and plated onto LB agar containing 40 µg of kanamycin per ml (for strains carrying pKT-kan), SS agar (for S. enterica strains), or SMAC agar (for E. coli strains) to determine the number of CFU per sprout. Tenfold dilutions of irrigation water samples were prepared in sterile water and plated onto the appropriate media to determine the number of CFU per milliliter of water.

Plasmid pKT-kan stability and GFP expression stability in S. enterica and E. coli strains were determined by plating dilutions of sprout homogenates on SS agar and SMAC agar, respectively, and determining the percentage of colonies emitting fluorescence under UV light. Over 1,000 colonies were examined for both Salmonella serovar Newport and E. coli O157:H7. These colonies were transferred from selective agar to LB agar plus kanamycin to determine the percentage of colonies that retained the plasmid but had lost GFP expression. Sprout homogenates were plated onto LB agar plus kanamycin, and several hundred resulting colonies were transferred to SS agar or SMAC agar, as appropriate, to determine if all green fluorescent colonies were S. enterica or E. coli O157:H7, as expected, or if the plasmid, pKT-kan, had transferred to other bacteria that might be present on the germinating alfalfa seeds.

Antibody labeling of Salmonella serovar Newport on alfalfa sprouts.
Alfalfa seeds were inoculated with Salmonella serovar Newport strain 96E01153C-TX as described above and sprouted for 2 days. Individual sprouts were removed from the water and incubated for 30 min in 1 ml of blocking buffer (1x PBS, 0.05% Tween 100, 1% bovine serum albumin fraction V). The blocking buffer was removed, 500 µl of anti-Salmonella lipopolysaccharide O:8 monoclonal antibody (obtained from Paul Duffey, California Department of Health, Berkeley) diluted 1:1,000 in blocking buffer was added, and the sprouts were incubated for 30 min. The sprouts were washed three times with 1 ml of blocking buffer and placed in 200 µl of a 1:50 dilution of fluorescein isothiocyanate-labeled rabbit anti-mouse immunoglobulin G antibody (Zymed Laboratories, Inc., San Francisco, Calif.) for 30 min. The sprouts were then washed three times with 1 ml of blocking buffer and observed with fluorescence microscopy. To determine if the antibody bound to the plant tissue, uninoculated alfalfa seeds were sanitized as described above, sprouted in sterile water, labeled with antibody as described above, and observed with fluorescence microscopy.

Microscopy.
Alfalfa seeds were inoculated with approximately 106 CFU of SeN(pKT) or Ec(pKT) per ml of water as described above and examined as they sprouted with a Leica MZ-FLIII fluorescence stereomicroscope (Leica Microsystems, Heildelberg, Germany) capable of magnifying samples 20- to 256-fold. For observation of green fluorescent bacteria on germinating sprouts, a 41017 Endow GFP filter set was used (Chroma Technology Corp., Brattleboro, Vt.). Images of the sprouting seeds were obtained with a Sony DKC-5000 digital camera (Sony Electronics, Inc., Tokyo, Japan). Images were imported into Adobe Photoshop (Adobe Systems, Inc., San Jose, Calif.) from a Sony DKS-5000 workstation.

The sprouting seeds were also examined by CLSM with a Leica TCS NT confocal microscope equipped with Ar, Kr, and He/Ne lasers (Leica Microsystems, Heildelberg, Germany). Images were obtained and modified by using Leica TCS NT software, version 2.0 (Leica Microsystems). In order to visualize sprout cells, the inoculated sprouts were stained with 20 mM propidium iodide (Molecular Probes, Eugene, Oreg.) diluted 1:200 in sterile water. An Ar laser (excitation wavelength, 488 nm) was used to excite the GFP, and a Kr laser (excitation wavelength, 568 nm) was used to excite the propidium iodide. GFP fluorescence was detected with a BP525/50 filter set and assigned the color green. Propidium iodide fluorescence was detected with a BP600/30 filter set and assigned the color red. Two color images were obtained by overlaying images from the green and red channels. All microscopy experiments were repeated at least three times, and multiple samples were examined each time.

Statistics.
Where indicated below, the plotted data are means and standard deviations of three to five measurements. Microsoft Excel 97 (Microsoft Corp., Redmond, Wash.) was used to perform Student's t tests and two-factor analysis of variance tests to determine the significance of the differences between the means.


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RESULTS AND DISCUSSION
 
S. enterica grows to higher levels on sprouting alfalfa seeds than E. coli O157:H7.
Alfalfa seeds were inoculated with Salmonella serovar Newport strain 96E01153C-TX, SeN(pKT), E. coli O157:H7 strain F4546, and Ec(pKT) and sprouted for 3 days, and samples were tested daily to determine if plasmid pKT-kan affected the growth of Salmonella serovar Newport strain 96E01153C-TX or E. coli O157:H7 strain F4546 on alfalfa sprouts (Fig. 1). There were no significant differences in the total numbers of bacteria on seeds or sprouts at any of the time points tested for Salmonella serovar Newport strain 96E01153C-TX with and without the plasmid or for E. coli O157:H7 strain F4546 with and without the plasmid (Student's t test; P > 0.05 for all time points tested). The plasmid was stable enough to examine where Salmonella serovar Newport and E. coli O157:H7 attached to the sprouts; 91% of SeN(pKT) colonies and 100% of Ec(pKT) colonies isolated from 2- and 3-day-old sprouts retained green fluorescence on SS agar and SMAC agar, respectively. Of the colonies on SS agar or SMAC agar, 100% of the SeN(pKT) colonies and 100% of the Ec(pKT) colonies were resistant to kanamycin; thus, the kanamycin resistance conferred by plasmid pKT-kan appears to be more stable than the GFP expression. All colonies isolated on LB agar plus kanamycin from samples inoculated with SeN(pKT) and from samples inoculated with EC(pKT) that were transferred to SS agar and SMAC agar formed colonies typical of S. enterica and E. coli O157:H7, respectively. Thus, there was no evidence that pKT-kan was transferred at a high rate to other bacteria that might have been present on the alfalfa sprouts.



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  		FIG. 1. Salmonella serovar Newport strain 96E01153C-TX, SeN(pKT), E. coli O157:H7 strain F4546, and Ec(pKT) levels on sprouting alfalfa seeds. The data points represent the means and standard deviations of the bacterial levels on inoculated seeds, 1-day-old sprouts, and 2-day-old sprouts. The experiment was repeated at least three times for each strain. Each experiment had three internal replicates, and generally, three sprout samples were taken from each replicate. Symbols: {blacklozenge}, Salmonella serovar Newport strain 96E01153C-TX; {lozenge}, SeN(pKT); •, E. coli O157:H7 strain F4546; {circ}, Ec(pKT).

While examining the effects of pKT-kan on Salmonella serovar Newport strain 96E01153C-TX and E. coli O157:H7 strain F4546 growth on sprouts, we noted that the Salmonella serovar Newport strain grew to higher levels on alfalfa sprouts than the E. coli O157:H7 strain (Fig. 1). We decided to test additional S. enterica and E. coli O157:H7 strains to determine if S. enterica strains grow to higher levels on sprouting seeds than E. coli O157:H7 strains. Four additional S. enterica strains representing four different serovars and five additional E. coli O157:H7 strains representing different sources were transformed with pKT-kan, and the growth of these strains on sprouting alfalfa seeds was examined (see Table 1 for strain details). The S. enterica strains reached significantly higher levels than the E. coli O157:H7 strains on the alfalfa sprouts over 2 days of sprout germination (Fig. 2) (Student's t test; P = 0.002). S. enterica may grow to higher levels on sprouting alfalfa seeds than E. coli O157:H7 because it is better able to utilize the sprout exudates and/or because it adheres better to the sprouts and is not washed away when the irrigation water is refreshed.



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  		FIG. 2. E. coli O157:H7 and S. enterica levels on 2-day-old alfalfa sprouts. The bars and lines represent the means and standard deviations of the changes in bacterial levels on inoculated seeds sprouted for 2 days. The experiment was repeated at least three times for each strain. Each experiment had three internal replicates, and generally, three sprout samples were taken from each replicate. The E. coli O157:H7 strains were as follows: A, F4546; B, 86-24; C, C7927; D, H2439; E, 96A13466; and F, EDL933. The S. enterica strains were as follows: G, S. enterica serovar Newport strain 96E01153C-TX; H, S. enterica serovar Mbandaka strain 99A 1668; I, S. enterica serovar Cubana strain 98A 9878; J, S. enterica serovar Havana strain 98A 4399; and K, S. enterica serovar Schwarzengrund strain 96E01152C-TX-1.

Sprouting temperature and inoculum dose affected the final levels of SeN(pKT) and Ec(pKT) on alfalfa sprouts and in alfalfa sprout irrigation water.
Alfalfa seeds were inoculated with 1, 10, 102 103, or 104 CFU of SeN(pKT) or Ec(pKT) per ml and incubated at 20, 30, or 35°C for 2 days. The SeN(pKT) and Ec(pKT) levels on the sprouts and in the irrigation water were determined after 2 days of seed sprouting to examine if the sprouting temperature and the inoculum dose affected the bacterial levels (Fig. 3). Both the sprouting temperature and the initial inoculum dose significantly affected the number of SeN(pKT) or Ec(pKT) CFU in the irrigation water and on the sprouts (two-factor analysis of variance; P < 0.01 ).



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  		FIG. 3. Numbers of SeN(pKT) and Ec(pKT) CFU per milliliter of irrigation water and per sprout at different inoculum doses and different sprouting temperatures. The data are means from three repetitions. Each experiment had five internal replicates, and one sprout sample and one water sample were taken from each replicate.

Individual samples inoculated with low levels of bacteria varied greatly, especially at the lowest inoculum doses and sprouting temperatures. For example, the concentrations in irrigation water from seeds inoculated with 1 CFU of SeN(pKT) per ml and with 1 CFU of Ec(pKT) per ml and incubated at 20°C varied between 60 and 5,400 CFU/ml and between 20 and 136,000 CFU/ml, respectively. Sprout producers are currently required to test every crop of sprouts for both S. enterica and E. coli O157:H7 by sampling sprout irrigation water from 48-h-old sprouts (2). There is no consensus for how sensitive these tests must be, in part because it is not known what level of pathogen might be present in the irrigation water. Although human pathogens can multiply more than 4 log10 on sprouting seeds under ideal conditions, we have demonstrated that the pathogen levels are affected by multiple variables that may result in lower final pathogen levels. For example, in our assays, the concentrations in some samples inoculated with 1 CFU/ml of water increased to only 20 CFU/ml of water over 2 days, an increase of only 1.3 log10. Thus, tests used to determine if sprout lots are pathogen free need to be sensitive enough to detect low levels of pathogens.

We did not change the irrigation water daily in this set of experiments because we did not realize that it might be an important variable in the growth of E. coli O157:H7 and S. enterica in association with sprouting alfalfa seeds. We expected much lower growth of Ec(pKT) in these experiments, based on the low levels which we observed in our initial experiments (Fig. 1 and 2).

Frequency of changing the irrigation water affects the final levels of Ec(pKT) but not the final levels of SeN(pKT) on alfalfa sprouts and in alfalfa sprout irrigation water.
To determine if the frequency of changing the irrigation water affected pathogen levels, alfalfa seeds were inoculated with 103 CFU of SeN(pKT) or Ec(pKT) per ml and sprouted at 20°C, and the irrigation water was changed approximately every 10 h. The SeN(pKT) and Ec(pKT) levels in the irrigation water and on the sprouts were determined after 2 days of sprouting, 2 h after the irrigation water was replaced, to examine if frequent replacement of the irrigation water affected the bacterial levels (Fig. 4). Frequent replacement of the irrigation water significantly reduced the Ec(pKT) levels both in the water (Student's t test; P = 0.015) and on the sprouts (Student's t test; P = 0.04) compared to samples in which the water was not replaced. In contrast, frequent replacement of the irrigation water did not significantly reduce the SeN(pKT) levels in the water (Student's t test; P = 0.09) or on the sprouts (Student's t test; P = 0.18). When the water was changed frequently, the total number of CFU of Ec(pKT) per sprout was significantly less than the total number of CFU of SeN(pKT) per sprout (Student's t test; P = 0.009).



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  		FIG. 4. Numbers of SeN(pKT) and Ec(pKT) CFU per ml of irrigation water and per sprout with and without regular irrigation water replacement. The data are means and standard deviations from three repetitions. Each experiment had five internal replicates, and one sprout sample and one water sample were taken from each replicate.

During production, sprouting seeds are frequently misted with water, and since E. coli O157:H7 levels are reduced by exchange of irrigation water, water replacement during seed sprouting is an important consideration for experimental modeling of sprouting conditions in production facilities. Since E. coli O157:H7 levels are significantly affected by irrigation water replacement, the rate and volume of irrigation that result in the lowest levels of E. coli O157:H7 on sprouts need to be determined. Also, since refreshing the irrigation water reduces E. coli O157:H7 populations, when seed samples are sprouted to test for this pathogen, the irrigation water should not be changed since this significantly reduces the population of E. coli O157:H7 on the sprouts.

Attachment patterns of SeN(pKT) and Ec(pKT) on sprouting alfalfa seeds.
Seeds were inoculated with SeN(pKT) and Ec(pKT), sprouted for 2 to 3 days, and observed daily with fluorescence stereomicroscopy to determine where these strains colonize sprouting seeds (Fig. 5). Alfalfa seed autofluorescence and the low levels of bacteria present inhibited observation of bacteria on seeds for several hours after inoculation. SeN(pKT) colonized the broken edges of the sprout seed coats during the first 24 h of seed germination (Fig. 5A), while in contrast, Ec(pKT) colonized the emerging radicle along the root cell junctions (Fig. 5B). By the second day of sprouting, large SeN(pKT) aggregates were visible on the region of the alfalfa roots where the root hairs developed (Fig. 5C). Ec(pKT) colonized the same region, but bacterial aggregates were not observed (Fig. 5D). The SeN(pKT) and Ec(pKT) attachment patterns for 3-day-old sprouts resembled those for 2-day-old sprouts. SeN(pKT) and Ec(pKT) cells were visible only rarely on the hypocotyls or cotyledons of the 2- and 3-day-old sprouts.



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  		FIG. 5. Fluorescence stereoscope micrographs of SeN(pKT) and Ec(pKT) on sprouting alfalfa seeds. (A) SeN(pKT)-colonized seed coat edge of 1-day-old alfalfa sprout. (B) Ec(pKT)-colonized radicle of 1-day-old alfalfa sprout. (C) SeN(pKT)-colonized root in the region where the root hairs develop on 2-day-old alfalfa sprouts. The clumpy appearance of the GFP-labeled cells on the sprout is due to the large SeN(pKT) aggregates on the sprout. (D) Ec(pKT)-colonized root in the region where the root hairs develop on 2-day-old alfalfa sprouts. Cell aggregates are not visible. sc, seed coat; ra, radicle; r, root.

Seed coats and 2-day-old sprouts grown from seeds inoculated with SeN(pKT) or Ec(pKT) were stained with propidium iodide so that the alfalfa root and seed coat cells fluoresced, and the sprouts were examined by CSLM (Fig. 6). SeN(pKT) aggregates were observed on the sprout roots, surrounding the base and at the tips of the sprout root hairs, and on the broken edges of the seed coats (Fig. 6A and B). Ec(pKT) cells were observed in the same locations but in much lower numbers and as single cells rather than as cell aggregates (Fig. 6C and D). Individual SeN(pKT) and Ec(pKT) cells were observed occasionally on the cotyledons and the hypocotyls.



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  		FIG. 6. CLSM micrographs of SeN(pKT) and Ec(pKT) on sprouting alfalfa seeds. (A) SeN(pKT) aggregates on the root and on the root hairs of 2- or 3-day-old alfalfa sprout. (B) SeN(pKT) aggregates on alfalfa seed coat edge. (C) Individual Ec(pKT) cells on alfalfa root. (D) Individual Ec(pKT) cells on alfalfa seed coat edge. rh, root hair; sc, seed coat.

To confirm the results obtained with GFP-labeled bacteria, we used anti-Salmonella lipopolysaccharide O:8 monoclonal antibody in conjunction with fluorescein isothiocyanate-labeled anti-mouse antibody to detect Salmonella serovar Newport strain 96E01153C-TX on inoculated sprouts. We were able to detect Salmonella serovar Newport strain 96E01153C-TX present on the seed coat edges and roots of the inoculated alfalfa sprouts (data not shown), confirming that Salmonella serovar Newport strain 96E01153C-TX colonized the sprouts in a similar pattern whether it carried pKT-kan or not. The antibody did not bind to the seed coats or roots of uninoculated controls. The antibody labeling technique required multiple washes, yet Salmonella serovar Newport cells were still detected on seed coat edges and roots, suggesting that the cells were tightly associated with the sprouts.

Overall, our results demonstrate that both E. coli O157:H7 and S. enterica grow in association with sprouting alfalfa seeds and that the levels of these pathogens are affected by the initial inoculum dose, by the incubation temperature, and, for E. coli O157:H7, by the frequency of irrigation water exchange. We demonstrated for the first time that S. enterica strains grow and reach higher levels on sprouts than E. coli O157:H7 strains. These results, combined with our microscopy observations, confirm that S. enterica and E. coli O157:H7 may both grow in association with sprouting seeds but that S. enterica appears to adhere to alfalfa roots and seed coats significantly better than E. coli O157:H7.

A recent review of sprout-associated outbreaks reported that 80% of the outbreaks in the United States were caused by S. enterica (2), and all of the reported outbreaks associated with sprouts since this review was published have been caused by S. enterica. The difference in the ability to grow in association with sprouting seeds may partially explain why more outbreaks associated with fresh produce are caused by S. enterica than by E. coli O157:H7.


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ACKNOWLEDGMENTS
 
We thank Sharon Abbott, Paul Duffey, William Miller, and Marian Wachtel for providing strains, plasmids, and antibodies. We also thank Marian Wachtel, William Miller, Brion Duffy, Maria Brandl, and Anna Bates for helpful discussions and advice.

This research was funded by United States Department of Agriculture Agricultural Research Service CRIS project 5325-42000-022.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Plant Pathology, University of Wisconsin—Madison, 1630 Linden Dr., Madison, WI 53706. Phone: (608) 262-7911. Fax: (608) 263-2626. E-mail: amyc{at}plantpath.wisc.edu. Back


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REFERENCES
 
    1
  1. Andrews, W. H., P. B. Mislivec, C. R. Wilson, V. R. Bruce, P. L. Poelma, R. Gibson, M. W. Trucksess, and K. Young. 1982. Microbial hazards associated with bean sprouting. J. Assoc. Off. Anal. Chem. 65:241-248.[Medline]
    2. 2
  2. Anonymous. 1999. Guidance for industry: reducing microbial food safety hazards for sprouted seeds and guidance for industry: sampling and microbial testing of spent irrigation water during sprout production. Fed. Regist. 64:57893-57902.
    3. 3
  3. Backer, H. D., J. C. Mohle-Boetani, S. B. Werner, S. L. Abbott, J. Farrar, and D. J. Vugia. 2000. High incidence of extra-intestinal infections in a Salmonella Havana outbreak associated with alfalfa sprouts. Public Health Rep. 115:339-345.
    4. 4
  4. Beuchat, L. R. 1997. Comparison of chemical treatments to kill Salmonella on alfalfa seeds destined for sprout production. Int. J. Food Microbiol. 34:329-333.[CrossRef][Medline]
    5. 5
  5. Brooks, J. T., S. Y. Rowe, P. Shillam, D. M. Heltzel, S. B. Hunter, L. Slutsker, R. M. Hoekstra, and S. P. Luby. 2001. Salmonella typhimurium infections transmitted by chlorine-pretreated clover sprout seeds. Am. J. Epidemiol. 154:1020-1028.[Abstract/Free Full Text]
    6. 6
  6. Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802-805.[Abstract/Free Full Text]
    7. 7
  7. Charkowski, A. O., C. Z. Sarreal, and R. E. Mandrell. 2001. Wrinkled alfalfa seeds harbor more aerobic bacteria and are more difficult to sanitize than smooth seeds. J. Food Prot. 64:1292-1298.[Medline]
    8. 8
  8. Como-Sabetti, K., S. Reagan, S. Allaire, K. Parrott, C. M. Simonds, S. Hrabowy, B. Ritter, W. Hall, J. Altamirano, R. Martin, F. Downes, G. Jennings, R. Barrie, M. F. Dorman, N. Keon, M. Kucab, A. Al Shab, B. Robinson-Dunn, S. Dietrich, L. Moshur, L. Reese, J. Smith, K. Wilcox, J. Tilden, G. Wojtala, J. D. Park, M. Winnett, L. Petrilack, L. Vasquez, S. Jenkins, E. Barrett, M. Linn, D. Woolard, R. Hackler, H. Martin, D. McWilliams, B. Rouse, S. Willis, J. Rullan, J. Miller, G. S. Henderson, J. Pearson, J. Beers, R. Davis, and D. Saunders. 1997. Outbreaks of Escherichia coli O157:H7 infection associated with eating alfalfa sprouts-Michigan and Virginia, June-July 1997. Morb. Mortal. Wkly. Rep. 46:741-744.
    9. 9
  9. Gandhi, M., S. Golding, S. Yaron, and K. R. Matthews. 2001. Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts. J. Food Prot. 64:1891-1898.[Medline]
    10. 10
  10. Griffin, P. M., S. M. Ostroff, R. V. Tauxe, K. D. Greene, J. G. Wells, J. H. Lewis, and P. A. Blake. 1988. Illnesses associated with Escherichia coli O157:H7 infections. A broad clinical spectrum. Ann. Intern. Med. 109:705-712.
    11. 11
  11. Inami, G. B., and S. E. Moler. 1999. Detection and isolation of Salmonella from naturally contaminated alfalfa seeds following an outbreak investigation. J. Food Prot. 62:662-664.[Medline]
    12. 12
  12. Itoh, Y., Y. Sugita-Konishi, F. Kasuga, M. Iwaki, Y. Hara-Kudo, N. Saito, Y. Noguchi, H. Konuma, and S. Kumagai. 1998. Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts. Appl. Environ. Microbiol. 64:1532-1535.[Abstract/Free Full Text]
    13. 13
  13. Jaquette, C. B., L. R. Beuchat, and B. E. Mahon. 1996. Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage. App. Environ. Microbiol. 62:2212-2215.[Abstract]
    14. 14
  14. Lang, M. M., B. H. Ingham, and S. C. Ingham. 2000. Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting. Int. J. Microbiol. 58:73-82.
    15. 15
  15. Mahon, B. E., A. Ponka, W. N. Hall, K. Komatsu, S. E. Dietrich, A. Siitonen, G. Cage, P. S. Hayes, M. A. Lambert-Fair, N. H. Bean, P. M. Griffin, and L. Slutsker. 1997. An international outbreak of Salmonella infections caused by alfalfa sprouts grown from contaminated seeds. J. Infect. Dis. 175:876-882.[Medline]
    16. 16
  16. Miller, W. G., J. H. Leveau, and S. E. Lindow. 2000. Improved gfp and inaZ broad-host-range promoter-probe vectors. Mol. Plant-Microbe Interact. 13:1243-1250.[Medline]
    17. 17
  17. Mohle-Boetani, J. C., J. A. Farrar, S. B. Werner, D. Minassian, R. Bryant, S. Abbott, L. Slutsker, and D. J. Vugia. 2001. Escherichia coli O157 and Salmonella infections associated with sprouts in California, 1996-1998. Ann. Intern. Med. 135:239-247.[Abstract/Free Full Text]
    18. 18
  18. O'Brien, A. O., T. A. Lively, M. E. Chen, S. W. Rothman, and S. B. Formal. 1983. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (SHIGA) like cytotoxin. Lancet i:702.
    19. 19
  19. Ponka, A., Y. Andersson, A. Siitonen, B. de Jong, M. Jahkola, O. Haikala, A. Kuhmonen, and P. Pakkala. 1995. Salmonella in alfalfa sprouts. Lancet 345:462-463.[Medline]
    20. 20
  20. Puohiniemi, R., T. Heiskanen, and A. Siitonen. 1997. Molecular epidemiology of two international sprout-borne Salmonella outbreaks. J. Clin. Microbiol. 35:2487-2491.[Abstract]
    21. 21
  21. Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308:681-685.[Abstract]
    22. 22
  22. Stewart, D., K. Reineke, J. Ulaszek, T. Fu, and M. Tortorello. 2001. Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds. Lett. Appl. Microbiol. 33:95-99.[CrossRef][Medline]
    23. 23
  23. Stewart, D. S., K. F. Reineke, J. M. Ulaszek, and M. L. Tortorello. 2001. Growth of Salmonella during sprouting of alfalfa seeds associated with salmonellosis outbreaks. J. Food Prot. 64:618-622.[Medline]
    24. 24
  24. Taormina, P. J., and L. R. Beuchat. 1999. Behavior of enterohemorrhagic Escherichia coli O157:H7 on alfalfa sprouts during the sprouting process as influenced by treatments with various chemicals. J. Food Prot. 62:850-856.[Medline]
    25. 25
  25. Taormina, P. J., and L. R. Beuchat. 1999. Comparison of chemical treatments to eliminate enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds. J. Food Prot. 62:318-324.[Medline]
    26. 26
  26. Uljas, H. E., and S. C. Ingham. 1999. Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider Appl. Environ. Microbiol. 65:1924-1929.
    27. 27
  27. Van Beneden, C. A., W. E. Keene, R. A. Strang, D. H. Werker, A. S. King, B. Mahon, K. Hedberg, A. Bell, M. T. Kelly, V. K. Balan, W. R. MacKenzie, and D. Fleming. 1999. Multinational outbreak of Salmonella enterica serotype Newport infections due to contaminated alfalfa sprouts. JAMA 281:158-162.[Abstract/Free Full Text]

Applied and Environmental Microbiology, June 2002, p. 3114-3120, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.3114-3120.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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