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The impact of extreme weather events on Salmonella internalization in lettuce and green onion
Food Research International 45 (2012) 1118–1122
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
The impact of extreme weather events on Salmonella internalization in lettuce and green onion Chongtao Ge a, Cheonghoon Lee b, Jiyoung Lee a, b,⁎ a b
Department of Food Science & Technology, The Ohio State University, 375 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210, USA College of Public Health, Division of Environmental Health Sciences, The Ohio State University, 375 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210, USA
a r t i c l e
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Keywords: Salmonella Typhimurium Internalization Extreme weather events Fresh produce
a b s t r a c t Salmonella internalization is an important issue in raw vegetable consumption because washing usually cannot remove or inactivate the internalized pathogens effectively. In this study, the impact of extreme weather events, drought and heavy rains, caused by climate change on the internalization of Salmonella Typhimurium was investigated. Two leafy green fresh produce, iceberg lettuce and green onion were chosen. Rhizosphere soil inoculation was conducted to mimic the contamination routes via soil and then root uptake. Most internalized S. Typhimurium were found in lettuce leaves and in the root portions of green onion under all three irrigation conditions (optimal, drought, storm). In general, high concentration of soil inoculation facilitated the internalization level in both lettuce and green onion. Under extreme weather conditions, the internalization of S. Typhimurium in lettuce occurred when the soil was contaminated with a high level of bacteria (8–9 log colony forming unit (CFU)/g soil) and under these conditions, the internalization level was higher than the lettuce grown at the optimal water condition, except with 8 log CFU/g contamination (storm). Under drought, the results showed high variation, but the level of internalization of S. Typhimurium in lettuce increased by 16 times (1.21 log CFU/g) and 27 times (1.43 log CFU/g) compared to the optimally irrigated group when the soil was contaminated with 8 log and 9 log CFU/g soil, respectively. Ten-fold increased internalization was observed in the over-irrigated lettuce leaves when the soil was contaminated with 9 log CFU/g soil. The green onion samples showed ~ 4 log CFU/g green onion of S. Typhimurium internalization when exposed to high level of contamination (N 7 log CFU/g soil), which is a much higher internalization rate than the lettuce (average 2–3 log CFU/g). However, from the green onion experiments, no apparent patterns of water stress that affect on the levels on the Salmonella internalization were observed. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction It is estimated that foodborne diseases caused 5000 deaths, 325,000 hospitalizations and 76 million illnesses in the US annually. The reported microbial agents are mainly virulent Escherichia coli, Salmonella, Campylobacter, Shigella, Cyclospora, Cryptosporidium, Yersinia, Listeria, Bacillus, noroviruses and hepatitis A virus (Mead et al., 1999). Contamination of fresh produce has become a growing concern as the incidence of foodborne outbreak from fruits and vegetables increases and it is often consumed as raw (Gaynor et al., 2009; Hernández, Monge, Jiménez, & Taylor, 1997). Fresh produce production practices commonly include a washing step in which the produce are sanitized using water with 100 to 200 ppm of chlorine (Beuchat, Nail, Adler, & Clavero, 1998). The effectiveness of chlorine and other sanitizers is likely dependent on ⁎ Corresponding author at: College of Public Health, Division of Environmental Health Sciences, The Ohio State University, 375 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210, USA. Tel.: + 1 614 292 5546; fax: + 1 614 293 7710. E-mail address: [email protected] (J. Lee). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.06.054
whether the microorganisms are readily accessible. Thus, it is not able to fully inactivate the bacteria that have penetrated into the plant (Seo & Frank, 1999; Taormina, Beuchat, & Slutsker, 1999). Therefore, pathogen internalization within fresh produce has been a critical issue because it would preclude effective disinfection by washing and sanitizers (Sapers, 2001). Internalization can occur naturally due to contamination during growing in the field with improperly treated manure, runoff from livestock, wild animals, physical damage by insects or environmental factors (Beuchat, 2006; Erickson, Liao, et al., 2010), and irrigation with contaminated water (Beuchat et al., 1998; Strauch & Ballarini, 1994). In recent years, internalization of human pathogen in the edible aerial tissues of various fresh produce via root uptake has been widely reported (Bernstein, Sela, Pinto, & Ioffe, 2007; Guo, van Iersel, Chen, Brackett, & Beuchat, 2002; Solomon, Yaron, & Matthews, 2002). Microbial internalization within fruits and vegetables can also occur during packing or processing when a partial vacuum is applied (Bartz, 1982; Beuchanan, Edelson, Miller, & Sapers, 1999). Some foodborne pathogens, such as E. coli O157:H7 and Salmonella, have been found to be more resistant to the environmental changes from climate change (FAO, 2008). Salmonella is one of the most common
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human pathogens to cause foodborne outbreaks. According to Centers for Disease Control and Prevention (CDC, 2010), 8 multistate Salmonella outbreaks were reported during 2010, which is almost 3 times that of the previous year. A wide variety of food products can be contaminated with Salmonella, and the most susceptible commodities are fresh produce, chicken, shell eggs, peanut butter, and seafood (CDC, 2010). In this study, we focused on plant stress and its impact on Salmonella internalization in leafy green fresh produce, lettuce and green onion that are usually consumed raw. Our hypothesis was that bacterial internalization in the fresh produce could be influenced by the level of stress that the plants endure during pre-harvest period because the protective responsive system of the plants against the microbial invasions is probably altered. For this, we investigated extreme weather events as an abiotic stress factor because they have become more frequent due to global climate change (Karl, Melillo, & Peterson, 2009). Among the extreme weather events, we focused extended dryness and heavy rain as water stress to plants. This study can provide valuable information on the impact of extreme weather events on fresh produce safety, especially pathogen internalization during pre-harvest stage.
The green fluorescence protein (GFP)-labeled S. Typhimurium (GFP-S) was incubated in Luria-Bertani (LB) broth supplemented with 100 μg/ml ampicillin at 37 °C. After centrifugation at 8000 rpm for 10 min, bacteria were resuspended with 60 ml phosphate buffered saline and then adjusted to ~1011 CFU/ml. The cell density was determined using WPA cell density meter (WPA Biowave, UK) and was confirmed with a plate count method. Each batch of the plants was contaminated with 10 7, 108 and 109 CFU/g soil with the GFP- S suspension at the rhizosphere. The inoculums were prepared by making a final volume of 10–15 ml before inoculation (Bernstein, Sela, & NederLavon, 2007a). After inoculation, the plants inoculated with same concentration of S. Typhimurium were divided into three groups (each group contained two lettuce or green onion shoots) and were further grown in the lab for 2 days using 3 irrigation types with different watering volume as mentioned previously. Three lettuce and three green onion groups, without S. Typhimurium inoculated in the soil and growing under 3 different irrigation types respectively, were used as control groups. Plants were dug out after 2 days and the levels of S. Typhimurium internalization were determined with the method described below.
2. Materials and methods
2.3. Sample preparation and plate counts
2.1. Plant growth
Prior to the detection of the internalized salmonellae, the possible surviving S. Typhimurium on leaf surface was removed. After harvesting, the surface of whole plant was washed with tap water to fully remove the soil residues on the leaves or attached to the roots. Then the entire plants were disinfected with 80% ethanol for 5 s and then with 1% AgNO3 for 5 min (Franz et al., 2007). After rinsing with tap water and then deionized water (Milli-Q, Millipore, MA), all the plants were cut into 2 parts (leaves and roots), and then stomached for 2 min in a peptone water-filled sterile whirl-pak (NASCO, WI). The homogenized suspension (pH = ~ 7) of plant samples was directly spread onto LB agar supplemented with 100 μg/ml ampicillin. The soil samples from the rhizosphere inoculation experiments were diluted by peptone water to 10 − 3 and then plated on the ampicillin (100 μg/ml)-supplemented LB agar in duplicate, which were incubated at 37 °C overnight. Each set of the plant and soil sample was tested in duplicates, and all the experiments were repeated three times. The plates were examined under UV light (365 nm) to check if all the colonies on the plates still contained the recombinant plasmid and expressed GFP before numeration.
Iceberg lettuce (Lactuca sativa var. capitata) and green onion (Allium fistulosum) seeds were purchased from Burpee & Co. (Warmister, PA). Each plant was seeded in pots filled up with ~ 360 g sterile soil (Metro-Mix 360, SunGro Horticulture, Canada) and grown in a greenhouse (Department of Horticulture and Crop Science, The Ohio State University). The conditions of the mist room in the greenhouse were: 25–30 °C, 50–60% of relative humidity, overhead mist irrigation every 6 min, and ~ 16 h daylight per day. After germination, all the plants were transferred to a regular room in the greenhouse for manual irrigation. The cultivation condition was same as in the mist room, except 40–50% of relative humidity and irrigation volume of 500 ml/pot every 2 days for lettuce and 300 ml/pot every 2 days for green onion. After 4 weeks of growing, the plants were moved to a lab and kept in a growth chamber for S. Typhimurium inoculation. The lab cultivation conditions were: 23–27 °C, 30–45% of relative humidity, and ~16 hours lighting per day. In order to simulate water stress, the watering volume was explored and the plant growth condition was daily observed to determine the optimal irrigation volume and the water amount to lead to water stress (drought or storm) on the premise that the plants can still survive. To evaluate the actual water stress, the soil moisture content was determined using the gravimetric method (International Organization for Standardization (ISO), 1993). For lettuce samples, the optimal group was watered with 500 ml/pot for 2 days; the heavy rain (over-irrigation) group was watered with 1500 ml/pot for 2 days; for the drought group watering was stopped for the 2 days of post-inoculation. Different water volume was applied to the green onions; 300 ml was used for the optimal group; 1000 ml for the heavy rain (storm) group; no irrigation for the drought group within the following 2 days after inoculation. For the optimal and heavy rain (storm) groups, water was not directly poured onto the surface of soil. All the pots with holes at the bottom were placed into aluminum foil trays and the irrigation water was added into the trays immediately after the soil inoculation. This permitted the soil to uptake the water at different amounts while minimizing the dilution or drainage of the bacterial inoculums that might have occurred otherwise. 2.2. Soil contamination with Salmonella Salmonella Typhimurium (ATCC 19585) was labeled with pGFPuv plasmid (Clontech, CA) as described by Sambrook and Russell (2006).
2.4. Statistical analysis The data analysis was carried out with SPSS 17.0 statistical software (SPSS Inc., Chicago, IL). Analyses of variance were performed using Analysis of variance and Dunnett's test to determine the differences between the means of S. Typhimurium counts in each independent experiment, and soil moisture content changes. Results were considered significant at p b 0.05. 3. Results 3.1. Survival of S. Typhimurium in the rhizosphere soil Before S. Typhimurium contamination, the moisture content of soil was 2.20 ± 0.43 g/g dry soil (69% of wet weight). Under the optimal condition, the moisture content was 2.22 ± 0.32 g/g dry soil (69% of wet weight), which was not significantly changed until the end of the 2 days lab cultivation. The moisture content was 0.83 ± 0.41 g/g dry soil (45% of wet weight) under drought condition and 3.73 ± 0.11 g/g dry soil (79% of wet weight) until the end of 2 days post-inoculation. Both dryness (drought condition) and over irrigation (storm condition) significantly decreased and increased the moisture content in the soil, respectively (p b 0.05), which in turn changed the water
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condition of the plant growth after S. Typhimurium inoculation which changed the water condition of the plant growth during 2 days after S. Typhimurium inoculation. After the 2 days, the inoculated S. Typhimurium still largely survived in the soil (Table 1). However, a significant difference in the surviving S. Typhimurium between the extreme weather events and the optimal irrigation was not observed in the soil. In those 7 and 8 log CFU/g contamination group, the level of surviving Salmonella, which was still shedding in the rhizosphere soil, was 5–6 log CFU/gsoil. When the 9 log contamination group was checked, ~ 6.80 log CFU/g-soil of Salmonella was found in the rhizosphere soil. It seemed that the water stress did not affect the survival of Salmonella in the rhizosphere soil (p b 0.05).
3.2. Internalization of S. Typhimurium in the plants grown in contaminated soil under the water stress From the results of the plate counts, it was shown that S. Typhimurium was internalized in the lettuce and the green onion (Fig. 1). Especially, high concentration of S. Typhimurium (8–9 log CFU/g-soil) significantly increased their internalization in lettuce under the extreme irrigation conditions (Fig. 1). When the rhizosphere soil was contaminated with the level of 7 log CFU/g-soil of Salmonella, no internalization was detected in lettuce under both drought and storm conditions. Eight log CFU/g-soil inoculation led to 2.55 log CFU/g-lettuce of S. Typhimurium internalization under the drought conditions. At the level of 8 log CFU/g-soil Salmonella contamination, the internalized S. Typhimurium concentration was only 0.23 log CFU/g-lettuce and when the soil was inoculated with 9 log CFU/g, it increased to 3.53 log CFU/g-lettuce under the storm condition. In addition, we found that the internalization of S. Typhimurium increased under the water stress. When inoculated with 8 log CFU/g-soil, the drought group showed a significant increase of internalization in the lettuce than the optimal group (p=0.004). The storm group showed a significant increase in Salmonella internalization compare to the optimal group when the soil was inoculated with 9 log CFU/g of S. Typhimurium (p=0.001). In case of green onions, 8 log CFU/g-soil contamination events caused ~4 log CFU/g-green onion internalization, which was 1–3 log more than the green onions where were contaminated with 7 log CFU/g-soil under all the water stress conditions. Green onion had 0.57 log CFU/g-green onion of Salmonella when irrigated optimally, and the internalization level increased by 3.49 log under the drought condition and increased by 4.34 log under the storm condition when the soil was contaminated with 7 log CFU/g-soil. However, at the level of 9 log CFU/g-soil Salmonella contamination, the optimal group showed a significant increase in Salmonella internalization compare to the drought condition (p = 0.025) or the storm condition (p = 0.038).
Fig. 1. Internalized Salmonella in the whole lettuce and green onion through soil contamination growing under drought and heavy rain. S. Typhimurium was not detected in the plant samples which were grown in uncontaminated soil (control groups). The detection limit was 1.00 log CFU/g. When the sample yielded negative result, the number was assigned as 0.00.
3.3. Patterns of S. Typhimurium internalization between plant types and their body parts In the lettuce, the internalization of S. Typhimurium was only found in the leafy parts irrespective of the water stress (Fig. 2). In case of the green onion, however, the root samples showed a significantly higher internalization level than the leafy parts except those inoculated with 10 7 CFU/g soil of S. Typhimurium under the drought condition (Fig. 3). Under the optimal or the storm conditions, the internalized salmonellae increased in the leafy portion as the inoculated S. Typhimurium increased. But under the drought conditions, 10 9 CFU/g inoculums only resulted in 2.18 log CFU/g internalization, which was lower than samples inoculated with 10 8 CFU/g soil inoculation. The influence of water stress on S. Typhimurium internalization in green onion roots grown under extreme watering conditions exhibited no apparent patterns when the soil was heavily contaminated (8 or 9 log CFU/g-soil). In contrast, the water stress significantly enhanced the internalization in the roots as well as leaves when the soil was contaminated with lower level of Salmonella (7 log CFU/g). 4. Discussion The internalization of S. Typhimurium increases the risks of the raw consumption of fruit and vegetables and could cause the outbreaks of foodborne diseases. In this study, the internalization of S. Typhimurium in the different fresh produces (lettuce and green
Table 1 The surviving S. Typhimurium in rhizosphere soil under three irrigation conditions by the end of day-2 after inoculation.a Contamination level Surviving Salmonella (log CFU/g soil) (Lettuce) (log CFU/g-soilb)
7 8 9 a
Surviving Salmonella (Green onion) (log CFU/g-soilb)
Optimal Drought Storm
Optimal Drought Storm
5.42 ± 0.60 5.72 ± 0.17 6.81 ± 0.03
5.48 ± 0.62 5.80 ± 0.31 6.80 ± 0.43
5.18 ± 0.62 5.62 ± 0.61 6.80 ± 0.09
5.04 ± 0.40 5.36 ± 0.41 6.80 ± 0.43
5.63 ± 0.32 5.59 ± 0.10 6.81 ± 0.18
5.11 ± 0.210 5.73 ± 0.29 6.73 ± 0.58
S. Typhimurium was not detected in all control groups. b Values are mean ± standard deviation for direct plate counts (log CFU per gram soil). The detection limit was 2.00 log CFU/g.
Fig. 2. Internalized S. Typhimurium detected in the separated portions of lettuce growing under drought and heavy rain. S. Typhimurium was not detected in the lettuce which was grown in uncontaminated soil (control groups). The detection limit was 1.00 log CFU/g. When the sample yielded negative result, the number was assigned as 0.00.
C. Ge et al. / Food Research International 45 (2012) 1118–1122
Fig. 3. Internalized S. Typhimurium detected in the separated portions of green onion growing under drought and heavy rain. S. Typhimurium was not detected in the green onion which was grown in uncontaminated soil (control groups). The detection limit was 1.00 log CFU/g. When the sample yielded negative result, the number was assigned as 0.00.
onion) grown under the water stress induced by extreme weather events was investigated. S. Typhimurium was inoculated in the rhizosphere soil to simulate dipping irrigation and the internalized S. Typhimurium in the different parts (leaf, root) was measured to track and explore the possible patterns of transport and fate of the internalization due to the water stress. Previously in our preliminary experiments, 10 6 CFU/g-soil of S. Typhimurium was inoculated at the rhizosphere before utilizing higher concentrations, but no internalization was detected in either plant (data not shown). When the inoculums were increased to 10 7 CFU/g-soil, Salmonella colonization within green onion and lettuce became detectable under all irrigation conditions (Fig. 3). Several researchers reported similar findings that lower concentration of human pathogens (b6 log CFU/g-soil) rarely internalized in the above ground tissue of plants in either field or lab studies (Erickson, Webb, et al., 2010b; Sharma et al., 2009). For example, Bernstein, Sela, and Neder-Lavon (2007b) observed internalization in the edible portion of 33-day-old lettuce after 2 days post-inoculation when the soil was contaminated with 8 log CFU/g of Salmonella enterica serovar Newport, while not detected in the 17-day-old lettuce. However, no internalization was found in both groups (17- and 33-day-old) when using 6 log CFU/g soil inoculation even though some samples were wounded to facilitate the internalization intentionally. It seems that a high concentration of human pathogens could create stress on plant root or there might be a concentration threshold, which varies in plant types and the growth condition as well, required to induce pathogen internalization in the plants or to initiate transport of the pathogens from the root to the edible above-ground portion. These things have not been fully elucidated yet. The confirmed fact was that the internalized S. Typhimurium, in either the edible portion or the root portions, was surely originated from the contaminated soil. The possible explanation is that the transmission of bacteria from soil to the root was a simply passive uptake through the root system, such as root junction and root tips (Solomon et al., 2002). In addition, the rhizosphere effect, which means rhizosphere had higher density of microbes than non-rhizosphere soil, seemed to be evident to play a key role in the bacterial internalization (Buyer, Roberts, & RussekCohen, 2002). In contrast, the chemotaxis study revealed that S. enterica serovars were able to actively move to the rhizosphere and then entered the root because the lettuce root exudates contained abundant nutrients, such as sugars, which they could be utilize as a carbon source by Salmonella (Klerks, Franz, van Gent-Pelzer, Zijlstra, & van Bruggen, 2007). The internalization results of the lettuce samples showed a dosedependent pattern under all the irrigation types (Fig. 1). Several investigators reported that drought can inhibit root growth and disturb
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the normal root functions, which could be a reason for inhibiting internalization in lettuce at a relatively lower level of contamination and increasing the threshold of S. Typhimurium internalization (Arkhipova et al., 2007; Werner et al., 2003). The internalization threshold was also as high as 108 CFU/g under the storm condition and this might be due to the damage to the roots when continuously being kept in the oversaturated soil. The excessive water can block oxygen and other gas transfer between the root and the atmosphere, and the shortage of oxygen would cause aggregation of minerals around the root, which can contribute to plant injury (Drew, 1997). Flooding also has been found to increase the permeability of root membranes, and cause more nutrients to leak into the rhizosphere. This will facilitate the growth of soilborne phytopathogens, and some could cause root rot and affect the normal root functions (Kirkpatrick, Rupe, & Rothrock, 2006). Meanwhile, no apparent dosedependent pattern was obtained from the green onion samples (Fig. 1). This implies that the internalization efficiency might vary in each green onion individual until the contamination level was reaching to a threshold that could achieve the Salmonella internalization. Our study showed that more internalized salmonellae seem to be transported further up to the leafy parts of the plant at a higher level of contamination, especially in lettuce. Internalized Salmonella were detected only in the leafy parts of the lettuce samples. This might be due to the result from its chemotaxis; the lettuce leaves had much more nutrients produced by photosynthesis that can benefit bacterial growth and multiplication than in the root area (Kroupitski et al., 2009). GFP-labeling technique has been used in several studies for enumeration and visualization of pathogen internalization (Bernstein, Sela, Pinto, & Ioffe, 2007; Erickson, Webb, et al., 2010a). However, there might be a possibility that the levels of internalized S. Typhimurium were underestimated in this study because the recombinant bacteria might have lost the plasmid in the absence of antibiotic pressure in the soil. Thus, the actual internalization level may be more severe than it was reported in this study. Overall, our data showed that S. Typhimurium was able to penetrate root and enter into the edible portion of lettuce and green onion from contaminated soils. We found the internalized levels were different according to the plant types (lettuce, green onion) and body parts (leaf, root). The extreme weather conditions (drought or storm) caused by climate change can facilitate the internalization of S. Typhimurium in lettuce when the soil was contaminated by high concentration of Salmonella. Larger scale of study is warranted to better understand the level of Salmonella internalization impacted by water stress factors and the difference between plant types. Recommendation for consumers and fresh produce industry is to remove unnecessary bottom parts from the edible portion of green onion, which may have higher level of internalized Salmonella, during food processing or preparation in order to prevent Salmonella-associated outbreaks. Acknowledgments This research was funded through a grant from the Food Innovation Center at the Ohio State University. We wish to thank Mr. Ed Nangle and Dr. David Gardner for their help in plant cultivation in the green house, and Dr. Matthew Kleinhenz for his advice about soil. References Arkhipova, T. N., Prinsen, E., Veselov, S. U., Martinenko, E. V., Melentiev, A. I., & Kudoyarova, G. R. (2007). Cytokinin producing bacteria enhance plant growth in drying soil. Plant and Soil, 292(1–2), 305–315. Bartz, J. (1982). Infiltration of tomatoes immersed at different temperatures to different depths in suspensions of Erwinia carotovora subsp. Carotovora. Plant Disease, 66, 302–305. Bernstein, N., Sela, S., & Neder-Lavon, S. (2007). Effecct of irrigation regimes on persistence of Salmonella enterica serovar Newport in small experimental pots designed for plant cultivation. Irrigation Science, 26, 1–8. Bernstein, N., Sela, S., & Neder-Lavon, S. (2007). Assessment of contamination potential of lettuce by Salmonella enterica serovar Newport added to the plant growing medium. Journal of Food Protection, 70, 1717–1722.
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The impact of extreme weather events on Salmonella internalization in lettuce and green onion Chongtao Ge a, Cheonghoon Lee b, Jiyoung Lee a, b,⁎ a b
Department of Food Science & Technology, The Ohio State University, 375 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210, USA College of Public Health, Division of Environmental Health Sciences, The Ohio State University, 375 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210, USA
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Keywords: Salmonella Typhimurium Internalization Extreme weather events Fresh produce
a b s t r a c t Salmonella internalization is an important issue in raw vegetable consumption because washing usually cannot remove or inactivate the internalized pathogens effectively. In this study, the impact of extreme weather events, drought and heavy rains, caused by climate change on the internalization of Salmonella Typhimurium was investigated. Two leafy green fresh produce, iceberg lettuce and green onion were chosen. Rhizosphere soil inoculation was conducted to mimic the contamination routes via soil and then root uptake. Most internalized S. Typhimurium were found in lettuce leaves and in the root portions of green onion under all three irrigation conditions (optimal, drought, storm). In general, high concentration of soil inoculation facilitated the internalization level in both lettuce and green onion. Under extreme weather conditions, the internalization of S. Typhimurium in lettuce occurred when the soil was contaminated with a high level of bacteria (8–9 log colony forming unit (CFU)/g soil) and under these conditions, the internalization level was higher than the lettuce grown at the optimal water condition, except with 8 log CFU/g contamination (storm). Under drought, the results showed high variation, but the level of internalization of S. Typhimurium in lettuce increased by 16 times (1.21 log CFU/g) and 27 times (1.43 log CFU/g) compared to the optimally irrigated group when the soil was contaminated with 8 log and 9 log CFU/g soil, respectively. Ten-fold increased internalization was observed in the over-irrigated lettuce leaves when the soil was contaminated with 9 log CFU/g soil. The green onion samples showed ~ 4 log CFU/g green onion of S. Typhimurium internalization when exposed to high level of contamination (N 7 log CFU/g soil), which is a much higher internalization rate than the lettuce (average 2–3 log CFU/g). However, from the green onion experiments, no apparent patterns of water stress that affect on the levels on the Salmonella internalization were observed. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction It is estimated that foodborne diseases caused 5000 deaths, 325,000 hospitalizations and 76 million illnesses in the US annually. The reported microbial agents are mainly virulent Escherichia coli, Salmonella, Campylobacter, Shigella, Cyclospora, Cryptosporidium, Yersinia, Listeria, Bacillus, noroviruses and hepatitis A virus (Mead et al., 1999). Contamination of fresh produce has become a growing concern as the incidence of foodborne outbreak from fruits and vegetables increases and it is often consumed as raw (Gaynor et al., 2009; Hernández, Monge, Jiménez, & Taylor, 1997). Fresh produce production practices commonly include a washing step in which the produce are sanitized using water with 100 to 200 ppm of chlorine (Beuchat, Nail, Adler, & Clavero, 1998). The effectiveness of chlorine and other sanitizers is likely dependent on ⁎ Corresponding author at: College of Public Health, Division of Environmental Health Sciences, The Ohio State University, 375 Howlett Hall, 2001 Fyffe Ct. Columbus, OH 43210, USA. Tel.: + 1 614 292 5546; fax: + 1 614 293 7710. E-mail address: [email protected] (J. Lee). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.06.054
whether the microorganisms are readily accessible. Thus, it is not able to fully inactivate the bacteria that have penetrated into the plant (Seo & Frank, 1999; Taormina, Beuchat, & Slutsker, 1999). Therefore, pathogen internalization within fresh produce has been a critical issue because it would preclude effective disinfection by washing and sanitizers (Sapers, 2001). Internalization can occur naturally due to contamination during growing in the field with improperly treated manure, runoff from livestock, wild animals, physical damage by insects or environmental factors (Beuchat, 2006; Erickson, Liao, et al., 2010), and irrigation with contaminated water (Beuchat et al., 1998; Strauch & Ballarini, 1994). In recent years, internalization of human pathogen in the edible aerial tissues of various fresh produce via root uptake has been widely reported (Bernstein, Sela, Pinto, & Ioffe, 2007; Guo, van Iersel, Chen, Brackett, & Beuchat, 2002; Solomon, Yaron, & Matthews, 2002). Microbial internalization within fruits and vegetables can also occur during packing or processing when a partial vacuum is applied (Bartz, 1982; Beuchanan, Edelson, Miller, & Sapers, 1999). Some foodborne pathogens, such as E. coli O157:H7 and Salmonella, have been found to be more resistant to the environmental changes from climate change (FAO, 2008). Salmonella is one of the most common
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human pathogens to cause foodborne outbreaks. According to Centers for Disease Control and Prevention (CDC, 2010), 8 multistate Salmonella outbreaks were reported during 2010, which is almost 3 times that of the previous year. A wide variety of food products can be contaminated with Salmonella, and the most susceptible commodities are fresh produce, chicken, shell eggs, peanut butter, and seafood (CDC, 2010). In this study, we focused on plant stress and its impact on Salmonella internalization in leafy green fresh produce, lettuce and green onion that are usually consumed raw. Our hypothesis was that bacterial internalization in the fresh produce could be influenced by the level of stress that the plants endure during pre-harvest period because the protective responsive system of the plants against the microbial invasions is probably altered. For this, we investigated extreme weather events as an abiotic stress factor because they have become more frequent due to global climate change (Karl, Melillo, & Peterson, 2009). Among the extreme weather events, we focused extended dryness and heavy rain as water stress to plants. This study can provide valuable information on the impact of extreme weather events on fresh produce safety, especially pathogen internalization during pre-harvest stage.
The green fluorescence protein (GFP)-labeled S. Typhimurium (GFP-S) was incubated in Luria-Bertani (LB) broth supplemented with 100 μg/ml ampicillin at 37 °C. After centrifugation at 8000 rpm for 10 min, bacteria were resuspended with 60 ml phosphate buffered saline and then adjusted to ~1011 CFU/ml. The cell density was determined using WPA cell density meter (WPA Biowave, UK) and was confirmed with a plate count method. Each batch of the plants was contaminated with 10 7, 108 and 109 CFU/g soil with the GFP- S suspension at the rhizosphere. The inoculums were prepared by making a final volume of 10–15 ml before inoculation (Bernstein, Sela, & NederLavon, 2007a). After inoculation, the plants inoculated with same concentration of S. Typhimurium were divided into three groups (each group contained two lettuce or green onion shoots) and were further grown in the lab for 2 days using 3 irrigation types with different watering volume as mentioned previously. Three lettuce and three green onion groups, without S. Typhimurium inoculated in the soil and growing under 3 different irrigation types respectively, were used as control groups. Plants were dug out after 2 days and the levels of S. Typhimurium internalization were determined with the method described below.
2. Materials and methods
2.3. Sample preparation and plate counts
2.1. Plant growth
Prior to the detection of the internalized salmonellae, the possible surviving S. Typhimurium on leaf surface was removed. After harvesting, the surface of whole plant was washed with tap water to fully remove the soil residues on the leaves or attached to the roots. Then the entire plants were disinfected with 80% ethanol for 5 s and then with 1% AgNO3 for 5 min (Franz et al., 2007). After rinsing with tap water and then deionized water (Milli-Q, Millipore, MA), all the plants were cut into 2 parts (leaves and roots), and then stomached for 2 min in a peptone water-filled sterile whirl-pak (NASCO, WI). The homogenized suspension (pH = ~ 7) of plant samples was directly spread onto LB agar supplemented with 100 μg/ml ampicillin. The soil samples from the rhizosphere inoculation experiments were diluted by peptone water to 10 − 3 and then plated on the ampicillin (100 μg/ml)-supplemented LB agar in duplicate, which were incubated at 37 °C overnight. Each set of the plant and soil sample was tested in duplicates, and all the experiments were repeated three times. The plates were examined under UV light (365 nm) to check if all the colonies on the plates still contained the recombinant plasmid and expressed GFP before numeration.
Iceberg lettuce (Lactuca sativa var. capitata) and green onion (Allium fistulosum) seeds were purchased from Burpee & Co. (Warmister, PA). Each plant was seeded in pots filled up with ~ 360 g sterile soil (Metro-Mix 360, SunGro Horticulture, Canada) and grown in a greenhouse (Department of Horticulture and Crop Science, The Ohio State University). The conditions of the mist room in the greenhouse were: 25–30 °C, 50–60% of relative humidity, overhead mist irrigation every 6 min, and ~ 16 h daylight per day. After germination, all the plants were transferred to a regular room in the greenhouse for manual irrigation. The cultivation condition was same as in the mist room, except 40–50% of relative humidity and irrigation volume of 500 ml/pot every 2 days for lettuce and 300 ml/pot every 2 days for green onion. After 4 weeks of growing, the plants were moved to a lab and kept in a growth chamber for S. Typhimurium inoculation. The lab cultivation conditions were: 23–27 °C, 30–45% of relative humidity, and ~16 hours lighting per day. In order to simulate water stress, the watering volume was explored and the plant growth condition was daily observed to determine the optimal irrigation volume and the water amount to lead to water stress (drought or storm) on the premise that the plants can still survive. To evaluate the actual water stress, the soil moisture content was determined using the gravimetric method (International Organization for Standardization (ISO), 1993). For lettuce samples, the optimal group was watered with 500 ml/pot for 2 days; the heavy rain (over-irrigation) group was watered with 1500 ml/pot for 2 days; for the drought group watering was stopped for the 2 days of post-inoculation. Different water volume was applied to the green onions; 300 ml was used for the optimal group; 1000 ml for the heavy rain (storm) group; no irrigation for the drought group within the following 2 days after inoculation. For the optimal and heavy rain (storm) groups, water was not directly poured onto the surface of soil. All the pots with holes at the bottom were placed into aluminum foil trays and the irrigation water was added into the trays immediately after the soil inoculation. This permitted the soil to uptake the water at different amounts while minimizing the dilution or drainage of the bacterial inoculums that might have occurred otherwise. 2.2. Soil contamination with Salmonella Salmonella Typhimurium (ATCC 19585) was labeled with pGFPuv plasmid (Clontech, CA) as described by Sambrook and Russell (2006).
2.4. Statistical analysis The data analysis was carried out with SPSS 17.0 statistical software (SPSS Inc., Chicago, IL). Analyses of variance were performed using Analysis of variance and Dunnett's test to determine the differences between the means of S. Typhimurium counts in each independent experiment, and soil moisture content changes. Results were considered significant at p b 0.05. 3. Results 3.1. Survival of S. Typhimurium in the rhizosphere soil Before S. Typhimurium contamination, the moisture content of soil was 2.20 ± 0.43 g/g dry soil (69% of wet weight). Under the optimal condition, the moisture content was 2.22 ± 0.32 g/g dry soil (69% of wet weight), which was not significantly changed until the end of the 2 days lab cultivation. The moisture content was 0.83 ± 0.41 g/g dry soil (45% of wet weight) under drought condition and 3.73 ± 0.11 g/g dry soil (79% of wet weight) until the end of 2 days post-inoculation. Both dryness (drought condition) and over irrigation (storm condition) significantly decreased and increased the moisture content in the soil, respectively (p b 0.05), which in turn changed the water
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condition of the plant growth after S. Typhimurium inoculation which changed the water condition of the plant growth during 2 days after S. Typhimurium inoculation. After the 2 days, the inoculated S. Typhimurium still largely survived in the soil (Table 1). However, a significant difference in the surviving S. Typhimurium between the extreme weather events and the optimal irrigation was not observed in the soil. In those 7 and 8 log CFU/g contamination group, the level of surviving Salmonella, which was still shedding in the rhizosphere soil, was 5–6 log CFU/gsoil. When the 9 log contamination group was checked, ~ 6.80 log CFU/g-soil of Salmonella was found in the rhizosphere soil. It seemed that the water stress did not affect the survival of Salmonella in the rhizosphere soil (p b 0.05).
3.2. Internalization of S. Typhimurium in the plants grown in contaminated soil under the water stress From the results of the plate counts, it was shown that S. Typhimurium was internalized in the lettuce and the green onion (Fig. 1). Especially, high concentration of S. Typhimurium (8–9 log CFU/g-soil) significantly increased their internalization in lettuce under the extreme irrigation conditions (Fig. 1). When the rhizosphere soil was contaminated with the level of 7 log CFU/g-soil of Salmonella, no internalization was detected in lettuce under both drought and storm conditions. Eight log CFU/g-soil inoculation led to 2.55 log CFU/g-lettuce of S. Typhimurium internalization under the drought conditions. At the level of 8 log CFU/g-soil Salmonella contamination, the internalized S. Typhimurium concentration was only 0.23 log CFU/g-lettuce and when the soil was inoculated with 9 log CFU/g, it increased to 3.53 log CFU/g-lettuce under the storm condition. In addition, we found that the internalization of S. Typhimurium increased under the water stress. When inoculated with 8 log CFU/g-soil, the drought group showed a significant increase of internalization in the lettuce than the optimal group (p=0.004). The storm group showed a significant increase in Salmonella internalization compare to the optimal group when the soil was inoculated with 9 log CFU/g of S. Typhimurium (p=0.001). In case of green onions, 8 log CFU/g-soil contamination events caused ~4 log CFU/g-green onion internalization, which was 1–3 log more than the green onions where were contaminated with 7 log CFU/g-soil under all the water stress conditions. Green onion had 0.57 log CFU/g-green onion of Salmonella when irrigated optimally, and the internalization level increased by 3.49 log under the drought condition and increased by 4.34 log under the storm condition when the soil was contaminated with 7 log CFU/g-soil. However, at the level of 9 log CFU/g-soil Salmonella contamination, the optimal group showed a significant increase in Salmonella internalization compare to the drought condition (p = 0.025) or the storm condition (p = 0.038).
Fig. 1. Internalized Salmonella in the whole lettuce and green onion through soil contamination growing under drought and heavy rain. S. Typhimurium was not detected in the plant samples which were grown in uncontaminated soil (control groups). The detection limit was 1.00 log CFU/g. When the sample yielded negative result, the number was assigned as 0.00.
3.3. Patterns of S. Typhimurium internalization between plant types and their body parts In the lettuce, the internalization of S. Typhimurium was only found in the leafy parts irrespective of the water stress (Fig. 2). In case of the green onion, however, the root samples showed a significantly higher internalization level than the leafy parts except those inoculated with 10 7 CFU/g soil of S. Typhimurium under the drought condition (Fig. 3). Under the optimal or the storm conditions, the internalized salmonellae increased in the leafy portion as the inoculated S. Typhimurium increased. But under the drought conditions, 10 9 CFU/g inoculums only resulted in 2.18 log CFU/g internalization, which was lower than samples inoculated with 10 8 CFU/g soil inoculation. The influence of water stress on S. Typhimurium internalization in green onion roots grown under extreme watering conditions exhibited no apparent patterns when the soil was heavily contaminated (8 or 9 log CFU/g-soil). In contrast, the water stress significantly enhanced the internalization in the roots as well as leaves when the soil was contaminated with lower level of Salmonella (7 log CFU/g). 4. Discussion The internalization of S. Typhimurium increases the risks of the raw consumption of fruit and vegetables and could cause the outbreaks of foodborne diseases. In this study, the internalization of S. Typhimurium in the different fresh produces (lettuce and green
Table 1 The surviving S. Typhimurium in rhizosphere soil under three irrigation conditions by the end of day-2 after inoculation.a Contamination level Surviving Salmonella (log CFU/g soil) (Lettuce) (log CFU/g-soilb)
7 8 9 a
Surviving Salmonella (Green onion) (log CFU/g-soilb)
Optimal Drought Storm
Optimal Drought Storm
5.42 ± 0.60 5.72 ± 0.17 6.81 ± 0.03
5.48 ± 0.62 5.80 ± 0.31 6.80 ± 0.43
5.18 ± 0.62 5.62 ± 0.61 6.80 ± 0.09
5.04 ± 0.40 5.36 ± 0.41 6.80 ± 0.43
5.63 ± 0.32 5.59 ± 0.10 6.81 ± 0.18
5.11 ± 0.210 5.73 ± 0.29 6.73 ± 0.58
S. Typhimurium was not detected in all control groups. b Values are mean ± standard deviation for direct plate counts (log CFU per gram soil). The detection limit was 2.00 log CFU/g.
Fig. 2. Internalized S. Typhimurium detected in the separated portions of lettuce growing under drought and heavy rain. S. Typhimurium was not detected in the lettuce which was grown in uncontaminated soil (control groups). The detection limit was 1.00 log CFU/g. When the sample yielded negative result, the number was assigned as 0.00.
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Fig. 3. Internalized S. Typhimurium detected in the separated portions of green onion growing under drought and heavy rain. S. Typhimurium was not detected in the green onion which was grown in uncontaminated soil (control groups). The detection limit was 1.00 log CFU/g. When the sample yielded negative result, the number was assigned as 0.00.
onion) grown under the water stress induced by extreme weather events was investigated. S. Typhimurium was inoculated in the rhizosphere soil to simulate dipping irrigation and the internalized S. Typhimurium in the different parts (leaf, root) was measured to track and explore the possible patterns of transport and fate of the internalization due to the water stress. Previously in our preliminary experiments, 10 6 CFU/g-soil of S. Typhimurium was inoculated at the rhizosphere before utilizing higher concentrations, but no internalization was detected in either plant (data not shown). When the inoculums were increased to 10 7 CFU/g-soil, Salmonella colonization within green onion and lettuce became detectable under all irrigation conditions (Fig. 3). Several researchers reported similar findings that lower concentration of human pathogens (b6 log CFU/g-soil) rarely internalized in the above ground tissue of plants in either field or lab studies (Erickson, Webb, et al., 2010b; Sharma et al., 2009). For example, Bernstein, Sela, and Neder-Lavon (2007b) observed internalization in the edible portion of 33-day-old lettuce after 2 days post-inoculation when the soil was contaminated with 8 log CFU/g of Salmonella enterica serovar Newport, while not detected in the 17-day-old lettuce. However, no internalization was found in both groups (17- and 33-day-old) when using 6 log CFU/g soil inoculation even though some samples were wounded to facilitate the internalization intentionally. It seems that a high concentration of human pathogens could create stress on plant root or there might be a concentration threshold, which varies in plant types and the growth condition as well, required to induce pathogen internalization in the plants or to initiate transport of the pathogens from the root to the edible above-ground portion. These things have not been fully elucidated yet. The confirmed fact was that the internalized S. Typhimurium, in either the edible portion or the root portions, was surely originated from the contaminated soil. The possible explanation is that the transmission of bacteria from soil to the root was a simply passive uptake through the root system, such as root junction and root tips (Solomon et al., 2002). In addition, the rhizosphere effect, which means rhizosphere had higher density of microbes than non-rhizosphere soil, seemed to be evident to play a key role in the bacterial internalization (Buyer, Roberts, & RussekCohen, 2002). In contrast, the chemotaxis study revealed that S. enterica serovars were able to actively move to the rhizosphere and then entered the root because the lettuce root exudates contained abundant nutrients, such as sugars, which they could be utilize as a carbon source by Salmonella (Klerks, Franz, van Gent-Pelzer, Zijlstra, & van Bruggen, 2007). The internalization results of the lettuce samples showed a dosedependent pattern under all the irrigation types (Fig. 1). Several investigators reported that drought can inhibit root growth and disturb
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the normal root functions, which could be a reason for inhibiting internalization in lettuce at a relatively lower level of contamination and increasing the threshold of S. Typhimurium internalization (Arkhipova et al., 2007; Werner et al., 2003). The internalization threshold was also as high as 108 CFU/g under the storm condition and this might be due to the damage to the roots when continuously being kept in the oversaturated soil. The excessive water can block oxygen and other gas transfer between the root and the atmosphere, and the shortage of oxygen would cause aggregation of minerals around the root, which can contribute to plant injury (Drew, 1997). Flooding also has been found to increase the permeability of root membranes, and cause more nutrients to leak into the rhizosphere. This will facilitate the growth of soilborne phytopathogens, and some could cause root rot and affect the normal root functions (Kirkpatrick, Rupe, & Rothrock, 2006). Meanwhile, no apparent dosedependent pattern was obtained from the green onion samples (Fig. 1). This implies that the internalization efficiency might vary in each green onion individual until the contamination level was reaching to a threshold that could achieve the Salmonella internalization. Our study showed that more internalized salmonellae seem to be transported further up to the leafy parts of the plant at a higher level of contamination, especially in lettuce. Internalized Salmonella were detected only in the leafy parts of the lettuce samples. This might be due to the result from its chemotaxis; the lettuce leaves had much more nutrients produced by photosynthesis that can benefit bacterial growth and multiplication than in the root area (Kroupitski et al., 2009). GFP-labeling technique has been used in several studies for enumeration and visualization of pathogen internalization (Bernstein, Sela, Pinto, & Ioffe, 2007; Erickson, Webb, et al., 2010a). However, there might be a possibility that the levels of internalized S. Typhimurium were underestimated in this study because the recombinant bacteria might have lost the plasmid in the absence of antibiotic pressure in the soil. Thus, the actual internalization level may be more severe than it was reported in this study. Overall, our data showed that S. Typhimurium was able to penetrate root and enter into the edible portion of lettuce and green onion from contaminated soils. We found the internalized levels were different according to the plant types (lettuce, green onion) and body parts (leaf, root). The extreme weather conditions (drought or storm) caused by climate change can facilitate the internalization of S. Typhimurium in lettuce when the soil was contaminated by high concentration of Salmonella. Larger scale of study is warranted to better understand the level of Salmonella internalization impacted by water stress factors and the difference between plant types. Recommendation for consumers and fresh produce industry is to remove unnecessary bottom parts from the edible portion of green onion, which may have higher level of internalized Salmonella, during food processing or preparation in order to prevent Salmonella-associated outbreaks. Acknowledgments This research was funded through a grant from the Food Innovation Center at the Ohio State University. We wish to thank Mr. Ed Nangle and Dr. David Gardner for their help in plant cultivation in the green house, and Dr. Matthew Kleinhenz for his advice about soil. References Arkhipova, T. N., Prinsen, E., Veselov, S. U., Martinenko, E. V., Melentiev, A. I., & Kudoyarova, G. R. (2007). Cytokinin producing bacteria enhance plant growth in drying soil. Plant and Soil, 292(1–2), 305–315. Bartz, J. (1982). Infiltration of tomatoes immersed at different temperatures to different depths in suspensions of Erwinia carotovora subsp. Carotovora. Plant Disease, 66, 302–305. Bernstein, N., Sela, S., & Neder-Lavon, S. (2007). Effecct of irrigation regimes on persistence of Salmonella enterica serovar Newport in small experimental pots designed for plant cultivation. Irrigation Science, 26, 1–8. Bernstein, N., Sela, S., & Neder-Lavon, S. (2007). Assessment of contamination potential of lettuce by Salmonella enterica serovar Newport added to the plant growing medium. Journal of Food Protection, 70, 1717–1722.
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