- Email: [email protected]
Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs
International Journal of Food Microbiology 145 (2011) 250–257
Contents lists available at ScienceDirect
International Journal of Food Microbiology 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 / i j f o o d m i c r o
Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs Dana Golberg a, Yulia Kroupitski a, Eduard Belausov b, Riky Pinto a, Shlomo Sela a,⁎ a Microbial Food-Safety Research Unit, Department of Food Quality & Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan 50250, Israel b Confocal Microscopy Unit, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan 50250, Israel
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 25 November 2010 Accepted 28 December 2010 Keywords: Salmonella Fresh produce Internalization Lettuce Leaf Stomata
a b s t r a c t Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (Pb 0.05). Internalization incidence in fresh basil was 46± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9± 3.3% and 0.56± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0–100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Enteric diseases linked to consumption of fresh produce have dramatically increased in the last several decades (Sivapalasingam et al., 2004; Tauxe et al., 1997). Fresh produce-associated outbreaks result in high economical losses to farmers, distributors and the food industry. Most outbreaks in the U.S. related to consumption of leafy greens were associated with Escherichia coli O157:H7, however, Salmonella enterica outbreaks related to this type of produce have also been reported (Hanning et al., 2009). S. enterica outbreaks linked to leafy greens were also reported in Europe (Raybaudi-Massilia et al., 2009) and included an outbreak of S. Senftenberg in the UK, Denmark and the Netherlands (Pezzoli et al., 2007), S. Thompson outbreak in Scandinavia and UK linked to consumption of rocket leaves (Nygard et al., 2008) and an S. Anatum infection associated with imported basil in Denmark (Pakalniskiene et al., 2009). These outbreaks underline the challenges to the agro-industrial sector as well as to the public health authorities. ⁎ Corresponding author. Microbial Food-Safety Research Unit, Department of Food Quality & Safety, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan, 50250, Israel. Tel.: +972 3 9683750; fax: +972 3 9683654. E-mail address: [email protected] (S. Sela). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.12.031
Fresh produce can become contaminated by human pathogens throughout the food-chain. It is accepted that plants might become contaminated in the field through the use of contaminated irrigation water and the use of animal manure for fertilization purposes (Beuchat and Ryu, 1997; Brandl, 2006; Franz and van Bruggen, 2008; Horby et al., 2003). Fresh produce can also become contaminated during harvest and at post-harvest stages due to poor workers' hygiene, and low sanitation in the processing plant (Beuchat and Ryu, 1997; Brandl, 2006). Studies on the interactions between E. coli O157:H7 and cut-lettuce leaves demonstrated efficient attachment of bacteria to the surface, trichomes, stomata, and cut edges (Seo and Frank, 1999; Takeuchi and Frank, 2000, 2001a,b). E. coli O157:H7 cells were occasionally observed to be entrapped 20 to 100 μm below the surface in stomata and cut-edges of iceberg lettuce (Seo and Frank, 1999). E. coli O157:H7 was also shown to colonize the inner tissues and stomata of cotyledons of radish sprouts, developed from seeds experimentally contaminated with the bacterium (Itoh et al., 1998). Recently, we have shown that Salmonella Typhimurium is capable of penetrating the epidermis of iceberg lettuce leaves through open stomata in a process that involves flagellar motility and chemotaxis (Kroupitski et al., 2009). In a recent study, E. coli O157:H7 was also shown to gain entry into internal tissue of baby spinach leaves. The internalization process required intact flagella
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
251
pre-conditioned for 20 min at high light intensity (100 μE m− 2 s− 1) at 30 °C, after pre-conditioning step 30 ml SDW was replaced with 30 ml Salmonella suspension (in final volume ca. 108 CFU/ml). Incubation proceeded for 2 h and the leaves were finally washed twice in SDW to remove unattached bacteria. GFP-labeled bacteria were visualized by a confocal laser-scanning microscope (Olympus IX81, Tokyo, Japan). Bacterial localization on leaf surface and underneath stomata was determined in 30 randomly chosen microscopic fields (magnification ×40), as described previously (Kroupitski et al., 2009). Since in many cases it was impossible to count individual bacteria, quantification of surface-associated and internalized bacteria was performed by calculating the percentage (incidence) of microscopic fields containing the indicated number of GFP-tagged bacteria (Table 1), divided by the total number of microscopic fields (30) examined in the same leaf and multiplied by 100. In order to facilitate the quantification of bacteria at the different sites, the data are presented in most cases as the incidence of Salmonella, i.e. percentage of fields (out of 30) containing ≥1 internal or surface-attached GFP-tagged bacteria. Each experiment was performed in triplicate (three different leaves of the same plant) and repeated at least four times at different days with different plants.
and type three secretion system (Xicohtencatl-Cortes et al., 2009). In contrast, Mitra et al. (2009) found no conclusive evidence for natural entry of this pathogen into the interior of spinach leaves. Since internalized bacteria are refractory to disinfection (Seo and Frank, 1999), bacterial internalization potentially poses a safety hazard to consumers. In order to assess the widespread stomatal internalization in leafy greens other than iceberg lettuce, we have examined the internalization of S. enterica sv. Typhimurium (S. Typhimurium) among several leafy vegetables and herbs. 2. Materials and methods 2.1. Bacterial strain and vegetables used in the study S. Typhimurium SL1344 strain expressing a green-fluorescent protein (Kroupitski et al., 2009) was used in this study. Internalization was examined in leaves of the following plants: lettuce (Lactuca sativa) (iceberg, romaine, and ruby red cultivars), arugula (Diplotaxis tenuifolia), parsley (Petroselinum crispum), basil (Ocimum basilicum L.), and tomato (Solanum lycopersicon cv MP1). All the vegetables, besides tomato leaves, were purchased at a local retail store and kept at 8 °C for 24 h before the onset of the experiments. Tomato leaves were aseptically cut from pot-grown plants and used immediately for the experiments.
2.3. Internalization survey in iceberg lettuce
2.2. Internalization assay
Incidence of internal Salmonella in iceberg lettuce leaves purchased between July 2008 and April 2010 was examined, as described previously (Section 2.2). Each month, four independent experiments (one in a week) with different lettuce heads were performed. Each experiment was performed in triplicate.
Bacterial growth and inoculation of leaves were performed essentially as described previously (Kroupitski et al., 2009). Briefly, bacteria were grown overnight in Luria–Bertani medium (LB) (Difco, Sparks, MD) at 37 °C, washed twice with sterile distilled water (SDW) and resuspended in SDW. Leaves of basil, iceberg-, romaine-, and red lettuce were aseptically cut into ca. 3 × 3 cm pieces using a sterile scalpel. In the case of parsley, arugula, and tomato, whole intact leaves of comparable fresh weight (0.5 g ± 0.1) were used. Leaves were submerged in 30 ml SDW in a 50 ml sterile polypropylene tube (Labcon, Petaluma, CA, USA), at one piece per tube. The leaves were
2.4. Scanning electron microscopy Lettuce leaf pieces incubated with Salmonella (ca. 108 CFU/ml) for 2 h at 30 °C were washed twice in phosphate-buffered saline (PBS), pH 7.2 and fixed in 5% glutaraldehyde in 0.1 M phosphate buffer for 2 h. The leaf pieces were washed and internal 2 × 2 mm squares were
Table 1 Distribution of Salmonella in leaf tissue of various leafy vegetables and fresh herbs. Number of microscope fields out of 30 (percentage) harboring Salmonella cellsa No. of cells per microscope field (×40)
0
1–10
10–50
50–100
≥100
Iceberg lettuce
Surface
0
Internal
5.7 ± 4.9 (19 ± 16) 0
7.6 ± 4.8 (25 ± 16) 6.7 ± 4.7 (22 ± 16) 10.1 ± 6.6 (33 ± 22) 1.3 ± 3.1 (4 ± 10) 16.9 ± 6.3 (56 ± 21) 1 ± 1.6 (3 ± 5) 0
3.8 ± 3.2 (12 ± 10) 1.7 ± 2.7 (6 ± 9) 1.5 ± 2.4 (5 ± 8) 0
0.7 ± 2 (2 ± 7) 1.1 ± 2.2 (4 ± 7) 0
24.1 ± 4.5 (80 ± 15) 0
18 ± 6.7 (60 ± 22) 14.4 ± 5.7 (48 ± 19) 18.4 ± 6.6 (61 ± 22) 3.4 ± 3.1 (11 ± 10) 2.2 ± 3.1 (7 ± 10) 4.5 ± 3.4 (15 ± 11) 0
7.3 ± 7.7 (24 ± 26) 0.3 ± 1 (1 ± 3) 0
3.6 ± 4.1 (12 ± 14) 0
8.7 ± 7.6 (29 ± 5) 0
8.6 ± 7.6 (29 ± 25) 0
2.6 ± 3.3 (9 ± 11) 0
29.4 ± 1 (98 ± 3) 0
0.6 ± 1 (2 ± 3) 2.8 ± 4.8 (9 ± 16) 11.3 ± 2.5 (38 ± 8) 4.2 ± 6.8 (14 ± 23) 0.2 ± 0.4 (0 ± 1)
0
0
13.6 ± 5.4 (45 ± 18) 2.2 ± 2.7 (7 ± 9) 14.8 ± 7.8 (49 ± 26) 0
9.3 ± 6.3 (31 ± 21) 0.4 ± 1 (1 ± 3) 9.7 ± 10 (32 ± 33) 0
Romaine lettuce
Surface Internal
Red lettuce
Surface Internal
Arugula
Surface Internal
Parsley
Surface Internal
Basil
Surface Internal
Tomato
Surface Internal
a
25.2 ± 4.8 (84 ± 16) 0
16.1 ± 3.6 (54 ± 12) 0 29.8 ± 0.4 (100 ± 1)
Each experiment was performed in triplicate (three different leaves of the same plant) and repeated at least four times at different days with different plants.
0 3.3 ± 3.8 (11 ± 13) 0 30 ± 0 (100 ± 0) 6.5 ± 11 (22 ± 37) 30 ± 0 (100 ± 0) 0 4.2 ± 7.6 (14 ± 25) 0 1.3 ± 2.8 (4 ± 9) 0
252
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
Fig. 1. Incidence of Salmonella in leaf surface and internal leaf tissues of various leafy vegetables and herbs. The incidence is calculated as the percentage of microscopic fields (×40) harboring ≥1 for internalized or attached Salmonella cell in 30 randomly chosen microscopic fields of leaf tissue. The data are the average of 4 independent experiments each performed in triplicates (3 × 30 fields per experiment). Different letters indicate significant difference (P b 0.05) according to ANOVA Tukey–Kramer Multiple Comparisons Test. A photograph of the tested leaves (not in scale) is presented at the bottom. White arrow indicates the leaf region that was examined.
excised, and processed for scanning electron microscopy as described previously (Kroupitski et al., 2009). Observation was performed under a Jeol JSM 35C-scanning electron microscope (Tokyo, Japan). Clusters of rod-shaped bacteria on the leaf surface were observed only in Salmonellainfected leaf samples but not in un-infected leaves (data not shown).
2.5. Statistical methods Statistical analysis of incidence of Salmonella in the different sites was performed by ANOVA Tukey–Kramer Multiple Comparisons Test using Instat version 3.0.6 (GraphPad Software, Inc., La Jolla, CA).
Fig. 2. Confocal microscopy images showing GFP-tagged bacteria residing on leaf surface and in internal leaf tissue following internalization assay. Internal leaf tissue images are composed of a stack of fluorescent images taken every 1.2 μm to a depth of 100 μm along a z section of the same field. All images were overlaid with differential interference contrast (DIC) images taken from the same location in each leaf. Bar denotes 50 μm.
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
3. Results 3.1. Localization of Salmonella on leaves from different vegetables The incidence of S. Typhimurium on leaf surface and underneath stomata is shown in Table 1 and in Fig. 1. Among the various leaves, iceberg lettuce and arugula demonstrate the highest incidence of internal Salmonella cells (81 ± 16% and 88 ± 16%, respectively; pre-
253
sented as the percentage of microscopic fields harboring ≥1 cells), while romaine, red-lettuce, and basil showed significantly (Pb 0.05) lower internalization incidence (16 ± 16%, 20± 15%, and 46 ± 12%, respectively). Parsley and tomato leaves show essentially no internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively), and the vast majority of Salmonella cells were confined to the leaf surface. Yet no apparent differences (PN 0.05) were found in the incidence of the pathogen on the leaves' surface (Fig. 1).
Fig. 3. Photomicrographs showing depth distribution of Salmonella cells in arugula leaf following internalization assay. (A) Representative photomicrographs showing fluorescent images along a z section overlaid with DIC images are shown. Red fluorescence indicates auto-fluorescence of chlorophyll within chloroplasts. Since, the epidermis is devoid of chloroplasts (besides the guard-cells), the presence of chloroplast (red) and nearby Salmonella (green) in the same focal plane confirms the localization of Salmonella cells within the parenchymal tissue. (B) A three-dimensional reconstruction of confocal microscopy images taken at the same leaf section shown above. The yellow color corresponds to the localization of bacteria (green) and chloroplast (red) in close proximity.
254
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
Confocal microscopy micrographs illustrating the distribution of Salmonella on the surface and in the leaf interior of arugula (high internalization incidence), basil (medium internalization incidence), and parsley (no internalization) are shown in Fig. 2. To confirm the internal localization of Salmonella in the leaf tissue, confocal microscopy images were photographed at various depths (z-sections). Representative images of S. Typhimurium in the substomatal space and in the intercellular region (apoplast) of the spongy parenchyma in arugula leaves (high internalization) are presented in Fig. 3A. A three-dimensional reconstruction of fluorescent images taken at the same leaf region further demonstrates the internal localization of the
GFP-tagged bacteria (Fig. 3B). In contrast to arugula, essentially no internalization occurred in parsley, and fluorescent Salmonella cells were only seen on the leaf surface (Fig. 4A and B). To explore the notion that anatomical differences may account for the lower internalization efficiency in some of the leaves, scanning electron microscopy was used to visualize the fine details of the stomata and their surrounding region (Fig. 5). Stomatal images were taken from leaves displaying high (arugula), medium (basil), and low (parsley) internalization efficiencies. Representing images show multiple bacteria localized near and on the rim of a stomata with no apparent structural differences among the leaves (Fig. 5). The majority of stomata in all the
Fig. 4. Photomicrographs showing depth distribution of Salmonella cells in parsley leaf following internalization assay. (A) GFP-labeled bacteria are observed only on the leaf surface. Red fluorescence indicates auto-fluorescence of chlorophyll within chloroplasts. (B) A three-dimensional reconstruction of confocal microscopy images taken at the same leaf section shown above demonstrates the existence of Salmonella cells (green) on the epidermis only.
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
255
Fig. 5. Scanning electron microscopy images showing the topography of a single stomate region in the different leaves with multiple bacteria (apparently Salmonella) located around. Bar denotes 10 μm.
leaves remained partially opened following infection, implying that differences in stomatal closure were not responsible for the diverse behavior. 3.2. Internalization efficiency varies throughout the year The internalization efficiency varied considerably throughout the year and between years (0–100%; Fig. 6). High internalization occurred mainly in the summer months of 2008–9 (Jul.–Sep.), with no or lower rate during the winter time (Dec. 2008, Feb. 2009, and Oct. 2009–Mar. 2010). Nevertheless, high incidence of internalization was also recorded on Jan. 2008 (winter). 4. Discussion
Incidence (%) of internal Salmonella
The ability of human pathogens to penetrate stomata and reside at sites inaccessible to sanitizers represents a potentially unique mode of contamination, which might lead to failure of standard washing and sanitation procedures to remove or inactivate pathogens on leafy vegetables. Microscopic evidences for the ability of various human pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis to enter into the leaf apoplast were previously reported (Jha et al., 2005; Plotnikova et al., 2000; Prithiviraj et al., 2005). Similarly, E. coli O157:H7 was observed in the leaf interior underneath stomata of iceberg lettuce (Seo and Frank, 1999; Takeuchi and Frank, 2000, 2001a,b). Recently, we have studied Salmonella internalization in iceberg lettuce and demonstrated that bacterial entry was dependent on motility and chemotaxis likely towards photosynthetic products (sugars) found in the apoplast underneath stomata (Kroupitski et al., 2009). Since, leaf internalization may potentially represent an impor-
tant mode of contamination, either in the field or during processing of fresh produce, we have examined in the present study the ability of Salmonella to internalize edible leaves of three different lettuce varieties, as well as leaves of fresh herbs, namely basil, parsley, arugula. In addition, internalization was studied in non-edible tomato leaves. Considerable internalization was observed in 5 of the 7 leaves, however, essentially no internalization was evident in the case of parsley and tomato leaves. Since, significant differences in the incidence of internalization were observed between closely-related lettuce cultivars, it is difficult to target any potential physiological, biochemical or morphological trait that might affect internalization. Unlike, iceberg lettuce (Kroupitski et al., 2009) and basil (this study), Salmonella displayed no specific attraction to stomata in arugula, parsley (Figs. 2–4), or tomato leaves (data not shown). Yet, the incidence of Salmonella in the apoplast of arugula and parsley differed considerably. In the case of arugula, it might be assumed that the pathogen is attracted similarly to both the cuticle and stomata and therefore multiple bacteria can be seen on various areas on the cuticle as well as underneath stomata. The lack of apparent internalization in the case of parsley might be explained by inhibition of bacterial motility and/or chemotaxis toward stomata, or by the lack of sufficient amount of chemoattractants in the sub-stomatal apoplast. Essential oils produced by basil are known for their antimicrobial activity against a range of bacteria including S. Typhimurium (Hammer et al., 1999). It is possible that the relatively lower incidence of internalization (46 ± 12%) in basil compared to iceberg lettuce and arugula was affected by these antibacterial agents. Bacterial colonization of the phyllosphere varies in different plants and is influenced by the surface properties of the leaf, including morphology, chemical constituents and metabolic activities (Beuchat, 2002; Heaton and Jones, 2008; Leveau, 2009; Lindow and Brandl, 2003;
100 80 60 40 20 0 Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr
2008
2009
2010
Fig. 6. Incidence of internal Salmonella in iceberg lettuce leaves purchased between July 2008 and April 2010. Data represent the mean ± SD of 4 independent experiments each in triplicate.
256
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
Yadav et al., 2005). SEM analysis revealed no obvious link between leaf morphology and the incidence of internalization, pointing perhaps to variations in the chemical properties of the tested leaves. An important feature of the results was the considerable variation in Salmonella internalization (0–100%) throughout the year and between years. High incidence of internalization was usually found in the summer months (July–September) in both 2008 and 2009. However, comparable internalization was observed also during Nov. and Dec. 2008, while essentially no internalization was found during the same months in 2009 (Fig. 6). It might be speculated that seasonal variations in day-length, light intensity, temperature, and humidity have influenced the content of chemoattractants in the leaves' apoplast and consequently caused variation in the incidence of internalization. In support of this notion, a seasonal difference in the microbial concentrations on leaves of fresh herbs has been reported. For example, in the case of cilantro and parsley, the concentrations of indicator bacteria were significantly higher in the fall than they were in spring or winter, and higher in spring than winter (Ailes et al., 2008). Similarly, seasonal conditions were shown to be an important factor in determining the fate of food-borne pathogens on fresh produce. Salmonella was detected at harvest on radish plants grown in contaminated field under conditions simulating wet and warm “summer” environment, but not under those simulating a comparatively drier and cooler “spring” environment (Natvig et al., 2002). Furthermore, competitiveness of Salmonella Thompson on wet cilantro leaves was significantly increased at plant incubation temperatures of 30 °C and 37 °C, enabling it to achieve higher population sizes than at 24 °C (Brandl and Mandrell, 2002). Although, higher internalization efficiencies were commonly recorded through summer months, a comparable high internalization incidence was also recorded once during a winter month (Jan. 2009). These findings, together with the month to month and year to year variations, suggest that other explanations, besides seasonal and climatic conditions might account for the different results. Since our study was carried on leafy vegetables that were purchased from a local supermarket; the identity of the iceberg lettuce variety is not known. It is possible that variation was simply related to differences in the lettuce cultivars used by farmers and sold in the supermarkets throughout the survey. A recent study, demonstrating different colonization efficiency in three spinach cultivars with different leaf surface morphologies (Mitra et al., 2009), further supports the importance of plant's cultivar in determining pathogen–plant interactions. Additional studies are required to clarify the relative role of seasonal parameters and the nature of the cultivar in the internalization process. 5. Conclusions Salmonella internalization seems to be variable among the seven different vegetables and fresh herbs examined in the present study; Two out of the seven (iceberg lettuce and arugula) displayed high incidence of internalization, 3 low to moderate (romaine and red lettuce, and basil, respectively), and 2 displayed very low incidence (parsley and tomato). Nonetheless, internalization of Salmonella in iceberg lettuce varied greatly (0–100%) from month to month and from year to year. Since internal Salmonella cells evade surface disinfection, it is highly important to assess leaf internalization in various leafy vegetables and fresh herbs. Identification of highly susceptible plants, as well as elucidation of plant- and environmentalfactors that affect internalization would be beneficial for the fresh produce sector and serve to better target its food safety resources. Acknowledgments This research was supported by Research Grant Award No. # US3949-06 from BARD, The United States - Israel Binational Agricultural
Research and Development Fund and by The Israeli Ministry of Agriculture, Chief scientist funds. English revision was done by Harriet Coleman.
References Ailes, E.C., Leon, J.S., Jaykus, L.A., Johnston, L.M., Clayton, H.A., Blanding, S., Kleinbaum, D.G., Backer, L.C., Moe, C.L., 2008. Microbial concentrations on fresh produce are affected by postharvest processing, importation, and season. Journal of Food Protection 71, 2389–2397. Beuchat, L.R., 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection 4, 413–423. Beuchat, L.R., Ryu, J.H., 1997. Produce handling and processing practices. Emerging Infectious Diseases 3, 459–465. Brandl, M.T., 2006. Fitness of human enteric pathogens on plants and implications for food safety. Annual Review of Phytopathology 44, 367–392. Brandl, M.T., Mandrell, R.E., 2002. Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere. Applied and Environmental Microbiology 68, 3614–3621. Franz, E., van Bruggen, A.H., 2008. Ecology of E. coli O157:H7 and Salmonella enterica in the primary vegetable production chain. Critical Reviews in Microbiology 34, 143–161. Hammer, K.A., Carson, C.F., Riley, T.V., 1999. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology 86, 985–990. Hanning, I.B., Nutt, J.D., Ricke, S.C., 2009. Salmonellosis outbreaks in the United States due to fresh produce: sources and potential intervention measures. Foodborne Pathogen Diseases 6, 635–648. Heaton, J.C., Jones, K., 2008. Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review. Journal of Applied Microbiology 104, 613–626. Horby, P.W., O'Brien, S.J., Adak, G.K., Graham, C., Hawker, J.I., Hunter, P., Lane, C., Lawson, A.J., Mitchell, R.T., Reacher, M.H., Threlfall, E.J., Ward, L.R., 2003. A national outbreak of multi-resistant Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 associated with consumption of lettuce. Epidemiology and Infection 130, 169–178. Itoh, Y., Sugita-Konishi, Y., Kasuga, F., Iwaki, M., Hara-Kudo, Y., Saito, N., Noguchi, Y., Konuma, H., Kumagai, S., 1998. Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts. Applied and Environmental Microbiology 64, 1532–1535. Jha, A.K., Bais, H.P., Vivanco, J.M., 2005. Enterococcus faecalis mammalian virulencerelated factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model. Infection and Immunity 73, 464–475. Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D., Sela, S., 2009. Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata. Applied and Environmental Microbiology 75, 6076–6086. Leveau, J.H.J., 2009. Microbiology: life on leaves. Nature 461, 741. Lindow, S.E., Brandl, M.T., 2003. Microbiology of the phyllosphere. Applied and Environmental Microbiology 69, 1875–1883. Mitra, R., Cuesta-Alonso, E., Wayadande, A., Talley, J., Gilliland, S., Fletcher, J., 2009. Effect of route of introduction and host cultivar on the colonization, internalization, and movement of the human pathogen Escherichia coli O157:H7 in spinach. Journal of Food Protection 72, 1521–1530. Natvig, E.E., Ingham, S.C., Ingham, B.H., Cooperband, L.R., Roper, T.R., 2002. Salmonella enterica serovar Typhimurium and Escherichia coli contamination of root and leaf vegetables grown in soils with incorporated bovine manure. Applied and Environmental Microbiology 68, 2737–2744. Nygard, K., Lassen, J., Vold, L., Andersson, Y., Fisher, I., Lofdahl, S., Threlfall, J., Luzzi, I., Peters, T., Hampton, M., Torpdahl, M., Kapperud, G., Aavitsland, P., 2008. Outbreak of Salmonella Thompson infections linked to imported rucola lettuce. Foodborne Pathogens and Disease 5, 165–173. Pakalniskiene, J., Falkenhorst, G., Lisby, M., Madsen, S.B., Olsen, K.E., Nielsen, E.M., Mygh, A., Boel, J., Molbak, K., 2009. A foodborne outbreak of enterotoxigenic E. coli and Salmonella Anatum infection after a high-school dinner in Denmark, November 2006. Epidemiology and Infection 137, 396–401. Pezzoli, L., Elson, R., Little, C., Fisher, I., Yip, H., Peters, T., Hampton, M., De Pinna, E., Coia, J.E., Mather, H.A., Brown, D.J., Nielsen, E.M., Ethelberg, S., Heck, M., de Jager, C., Threlfall, J., 2007. International outbreak of Salmonella Senftenberg in 2007. Eurosurveillance 12, E070614.3. Plotnikova, J.M., Rahme, L.G., Ausubel, F.M., 2000. Pathogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis. Plant Physiology 124, 1766–1774. Prithiviraj, B., Bais, H.P., Jha, A.K., Vivanco, J.M., 2005. Staphylococcus aureus pathogenicity on Arabidopsis thaliana is mediated either by a direct effect of salicylic acid on the pathogen or by SA-dependent, NPR1-independent host responses. The Plant Journal 42, 417–432. Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Martín-Belloso, O., 2009. Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. Comprehensive Reviews in Food Science and Food Safety 8, 157–180. Seo, K.H., Frank, J.F., 1999. Attachment of Escherichia coli O157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy. Journal of Food Protection 62, 3–9. Sivapalasingam, S., Friedman, C.R., Cohen, L., Tauxe, R.V., 2004. Fresh produce: growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. Journal of Food Protection 67, 2342–2353.
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257 Takeuchi, K., Frank, J.F., 2000. Penetration of Escherichia coli O157: H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. Journal of Food Protection 63, 434–440. Takeuchi, K., Frank, J.F., 2001a. Confocal microscopy and microbial viability detection for food research. Journal of Food Protection 64, 2088–2102. Takeuchi, K., Frank, J.F., 2001b. Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection. Journal of Food Protection 64, 147–151. Tauxe, R., Kruse, H., Hedberg, C., Potter, M., Madden, J., Wachsmuth, K., 1997. Microbial hazards and emerging issues associated with produce; a preliminary report to the
257
national advisory committee on microbiologic criteria for foods. Journal of Food Protection 60, 1400–1408. Xicohtencatl-Cortes, J., Sánchez Chacón, E., Saldaña, Z., Freer, E., Girón, J.A., 2009. Interaction of Escherichia coli O157:H7 with leafy green produce. Journal of Food Protection 72, 1531–1537. Yadav, R.K.P., Karamanoli, K., Vokou, D., 2005. Bacterial colonization of the phyllosphere of Mediterranean perennial species as influenced by leaf structure and chemical features. Microbial Ecology 50, 185–196.
Contents lists available at ScienceDirect
International Journal of Food Microbiology 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 / i j f o o d m i c r o
Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs Dana Golberg a, Yulia Kroupitski a, Eduard Belausov b, Riky Pinto a, Shlomo Sela a,⁎ a Microbial Food-Safety Research Unit, Department of Food Quality & Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan 50250, Israel b Confocal Microscopy Unit, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan 50250, Israel
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 25 November 2010 Accepted 28 December 2010 Keywords: Salmonella Fresh produce Internalization Lettuce Leaf Stomata
a b s t r a c t Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (Pb 0.05). Internalization incidence in fresh basil was 46± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9± 3.3% and 0.56± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0–100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Enteric diseases linked to consumption of fresh produce have dramatically increased in the last several decades (Sivapalasingam et al., 2004; Tauxe et al., 1997). Fresh produce-associated outbreaks result in high economical losses to farmers, distributors and the food industry. Most outbreaks in the U.S. related to consumption of leafy greens were associated with Escherichia coli O157:H7, however, Salmonella enterica outbreaks related to this type of produce have also been reported (Hanning et al., 2009). S. enterica outbreaks linked to leafy greens were also reported in Europe (Raybaudi-Massilia et al., 2009) and included an outbreak of S. Senftenberg in the UK, Denmark and the Netherlands (Pezzoli et al., 2007), S. Thompson outbreak in Scandinavia and UK linked to consumption of rocket leaves (Nygard et al., 2008) and an S. Anatum infection associated with imported basil in Denmark (Pakalniskiene et al., 2009). These outbreaks underline the challenges to the agro-industrial sector as well as to the public health authorities. ⁎ Corresponding author. Microbial Food-Safety Research Unit, Department of Food Quality & Safety, Agricultural Research Organization (ARO), The Volcani Center, POB 6, Beth-Dagan, 50250, Israel. Tel.: +972 3 9683750; fax: +972 3 9683654. E-mail address: [email protected] (S. Sela). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.12.031
Fresh produce can become contaminated by human pathogens throughout the food-chain. It is accepted that plants might become contaminated in the field through the use of contaminated irrigation water and the use of animal manure for fertilization purposes (Beuchat and Ryu, 1997; Brandl, 2006; Franz and van Bruggen, 2008; Horby et al., 2003). Fresh produce can also become contaminated during harvest and at post-harvest stages due to poor workers' hygiene, and low sanitation in the processing plant (Beuchat and Ryu, 1997; Brandl, 2006). Studies on the interactions between E. coli O157:H7 and cut-lettuce leaves demonstrated efficient attachment of bacteria to the surface, trichomes, stomata, and cut edges (Seo and Frank, 1999; Takeuchi and Frank, 2000, 2001a,b). E. coli O157:H7 cells were occasionally observed to be entrapped 20 to 100 μm below the surface in stomata and cut-edges of iceberg lettuce (Seo and Frank, 1999). E. coli O157:H7 was also shown to colonize the inner tissues and stomata of cotyledons of radish sprouts, developed from seeds experimentally contaminated with the bacterium (Itoh et al., 1998). Recently, we have shown that Salmonella Typhimurium is capable of penetrating the epidermis of iceberg lettuce leaves through open stomata in a process that involves flagellar motility and chemotaxis (Kroupitski et al., 2009). In a recent study, E. coli O157:H7 was also shown to gain entry into internal tissue of baby spinach leaves. The internalization process required intact flagella
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
251
pre-conditioned for 20 min at high light intensity (100 μE m− 2 s− 1) at 30 °C, after pre-conditioning step 30 ml SDW was replaced with 30 ml Salmonella suspension (in final volume ca. 108 CFU/ml). Incubation proceeded for 2 h and the leaves were finally washed twice in SDW to remove unattached bacteria. GFP-labeled bacteria were visualized by a confocal laser-scanning microscope (Olympus IX81, Tokyo, Japan). Bacterial localization on leaf surface and underneath stomata was determined in 30 randomly chosen microscopic fields (magnification ×40), as described previously (Kroupitski et al., 2009). Since in many cases it was impossible to count individual bacteria, quantification of surface-associated and internalized bacteria was performed by calculating the percentage (incidence) of microscopic fields containing the indicated number of GFP-tagged bacteria (Table 1), divided by the total number of microscopic fields (30) examined in the same leaf and multiplied by 100. In order to facilitate the quantification of bacteria at the different sites, the data are presented in most cases as the incidence of Salmonella, i.e. percentage of fields (out of 30) containing ≥1 internal or surface-attached GFP-tagged bacteria. Each experiment was performed in triplicate (three different leaves of the same plant) and repeated at least four times at different days with different plants.
and type three secretion system (Xicohtencatl-Cortes et al., 2009). In contrast, Mitra et al. (2009) found no conclusive evidence for natural entry of this pathogen into the interior of spinach leaves. Since internalized bacteria are refractory to disinfection (Seo and Frank, 1999), bacterial internalization potentially poses a safety hazard to consumers. In order to assess the widespread stomatal internalization in leafy greens other than iceberg lettuce, we have examined the internalization of S. enterica sv. Typhimurium (S. Typhimurium) among several leafy vegetables and herbs. 2. Materials and methods 2.1. Bacterial strain and vegetables used in the study S. Typhimurium SL1344 strain expressing a green-fluorescent protein (Kroupitski et al., 2009) was used in this study. Internalization was examined in leaves of the following plants: lettuce (Lactuca sativa) (iceberg, romaine, and ruby red cultivars), arugula (Diplotaxis tenuifolia), parsley (Petroselinum crispum), basil (Ocimum basilicum L.), and tomato (Solanum lycopersicon cv MP1). All the vegetables, besides tomato leaves, were purchased at a local retail store and kept at 8 °C for 24 h before the onset of the experiments. Tomato leaves were aseptically cut from pot-grown plants and used immediately for the experiments.
2.3. Internalization survey in iceberg lettuce
2.2. Internalization assay
Incidence of internal Salmonella in iceberg lettuce leaves purchased between July 2008 and April 2010 was examined, as described previously (Section 2.2). Each month, four independent experiments (one in a week) with different lettuce heads were performed. Each experiment was performed in triplicate.
Bacterial growth and inoculation of leaves were performed essentially as described previously (Kroupitski et al., 2009). Briefly, bacteria were grown overnight in Luria–Bertani medium (LB) (Difco, Sparks, MD) at 37 °C, washed twice with sterile distilled water (SDW) and resuspended in SDW. Leaves of basil, iceberg-, romaine-, and red lettuce were aseptically cut into ca. 3 × 3 cm pieces using a sterile scalpel. In the case of parsley, arugula, and tomato, whole intact leaves of comparable fresh weight (0.5 g ± 0.1) were used. Leaves were submerged in 30 ml SDW in a 50 ml sterile polypropylene tube (Labcon, Petaluma, CA, USA), at one piece per tube. The leaves were
2.4. Scanning electron microscopy Lettuce leaf pieces incubated with Salmonella (ca. 108 CFU/ml) for 2 h at 30 °C were washed twice in phosphate-buffered saline (PBS), pH 7.2 and fixed in 5% glutaraldehyde in 0.1 M phosphate buffer for 2 h. The leaf pieces were washed and internal 2 × 2 mm squares were
Table 1 Distribution of Salmonella in leaf tissue of various leafy vegetables and fresh herbs. Number of microscope fields out of 30 (percentage) harboring Salmonella cellsa No. of cells per microscope field (×40)
0
1–10
10–50
50–100
≥100
Iceberg lettuce
Surface
0
Internal
5.7 ± 4.9 (19 ± 16) 0
7.6 ± 4.8 (25 ± 16) 6.7 ± 4.7 (22 ± 16) 10.1 ± 6.6 (33 ± 22) 1.3 ± 3.1 (4 ± 10) 16.9 ± 6.3 (56 ± 21) 1 ± 1.6 (3 ± 5) 0
3.8 ± 3.2 (12 ± 10) 1.7 ± 2.7 (6 ± 9) 1.5 ± 2.4 (5 ± 8) 0
0.7 ± 2 (2 ± 7) 1.1 ± 2.2 (4 ± 7) 0
24.1 ± 4.5 (80 ± 15) 0
18 ± 6.7 (60 ± 22) 14.4 ± 5.7 (48 ± 19) 18.4 ± 6.6 (61 ± 22) 3.4 ± 3.1 (11 ± 10) 2.2 ± 3.1 (7 ± 10) 4.5 ± 3.4 (15 ± 11) 0
7.3 ± 7.7 (24 ± 26) 0.3 ± 1 (1 ± 3) 0
3.6 ± 4.1 (12 ± 14) 0
8.7 ± 7.6 (29 ± 5) 0
8.6 ± 7.6 (29 ± 25) 0
2.6 ± 3.3 (9 ± 11) 0
29.4 ± 1 (98 ± 3) 0
0.6 ± 1 (2 ± 3) 2.8 ± 4.8 (9 ± 16) 11.3 ± 2.5 (38 ± 8) 4.2 ± 6.8 (14 ± 23) 0.2 ± 0.4 (0 ± 1)
0
0
13.6 ± 5.4 (45 ± 18) 2.2 ± 2.7 (7 ± 9) 14.8 ± 7.8 (49 ± 26) 0
9.3 ± 6.3 (31 ± 21) 0.4 ± 1 (1 ± 3) 9.7 ± 10 (32 ± 33) 0
Romaine lettuce
Surface Internal
Red lettuce
Surface Internal
Arugula
Surface Internal
Parsley
Surface Internal
Basil
Surface Internal
Tomato
Surface Internal
a
25.2 ± 4.8 (84 ± 16) 0
16.1 ± 3.6 (54 ± 12) 0 29.8 ± 0.4 (100 ± 1)
Each experiment was performed in triplicate (three different leaves of the same plant) and repeated at least four times at different days with different plants.
0 3.3 ± 3.8 (11 ± 13) 0 30 ± 0 (100 ± 0) 6.5 ± 11 (22 ± 37) 30 ± 0 (100 ± 0) 0 4.2 ± 7.6 (14 ± 25) 0 1.3 ± 2.8 (4 ± 9) 0
252
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
Fig. 1. Incidence of Salmonella in leaf surface and internal leaf tissues of various leafy vegetables and herbs. The incidence is calculated as the percentage of microscopic fields (×40) harboring ≥1 for internalized or attached Salmonella cell in 30 randomly chosen microscopic fields of leaf tissue. The data are the average of 4 independent experiments each performed in triplicates (3 × 30 fields per experiment). Different letters indicate significant difference (P b 0.05) according to ANOVA Tukey–Kramer Multiple Comparisons Test. A photograph of the tested leaves (not in scale) is presented at the bottom. White arrow indicates the leaf region that was examined.
excised, and processed for scanning electron microscopy as described previously (Kroupitski et al., 2009). Observation was performed under a Jeol JSM 35C-scanning electron microscope (Tokyo, Japan). Clusters of rod-shaped bacteria on the leaf surface were observed only in Salmonellainfected leaf samples but not in un-infected leaves (data not shown).
2.5. Statistical methods Statistical analysis of incidence of Salmonella in the different sites was performed by ANOVA Tukey–Kramer Multiple Comparisons Test using Instat version 3.0.6 (GraphPad Software, Inc., La Jolla, CA).
Fig. 2. Confocal microscopy images showing GFP-tagged bacteria residing on leaf surface and in internal leaf tissue following internalization assay. Internal leaf tissue images are composed of a stack of fluorescent images taken every 1.2 μm to a depth of 100 μm along a z section of the same field. All images were overlaid with differential interference contrast (DIC) images taken from the same location in each leaf. Bar denotes 50 μm.
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
3. Results 3.1. Localization of Salmonella on leaves from different vegetables The incidence of S. Typhimurium on leaf surface and underneath stomata is shown in Table 1 and in Fig. 1. Among the various leaves, iceberg lettuce and arugula demonstrate the highest incidence of internal Salmonella cells (81 ± 16% and 88 ± 16%, respectively; pre-
253
sented as the percentage of microscopic fields harboring ≥1 cells), while romaine, red-lettuce, and basil showed significantly (Pb 0.05) lower internalization incidence (16 ± 16%, 20± 15%, and 46 ± 12%, respectively). Parsley and tomato leaves show essentially no internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively), and the vast majority of Salmonella cells were confined to the leaf surface. Yet no apparent differences (PN 0.05) were found in the incidence of the pathogen on the leaves' surface (Fig. 1).
Fig. 3. Photomicrographs showing depth distribution of Salmonella cells in arugula leaf following internalization assay. (A) Representative photomicrographs showing fluorescent images along a z section overlaid with DIC images are shown. Red fluorescence indicates auto-fluorescence of chlorophyll within chloroplasts. Since, the epidermis is devoid of chloroplasts (besides the guard-cells), the presence of chloroplast (red) and nearby Salmonella (green) in the same focal plane confirms the localization of Salmonella cells within the parenchymal tissue. (B) A three-dimensional reconstruction of confocal microscopy images taken at the same leaf section shown above. The yellow color corresponds to the localization of bacteria (green) and chloroplast (red) in close proximity.
254
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
Confocal microscopy micrographs illustrating the distribution of Salmonella on the surface and in the leaf interior of arugula (high internalization incidence), basil (medium internalization incidence), and parsley (no internalization) are shown in Fig. 2. To confirm the internal localization of Salmonella in the leaf tissue, confocal microscopy images were photographed at various depths (z-sections). Representative images of S. Typhimurium in the substomatal space and in the intercellular region (apoplast) of the spongy parenchyma in arugula leaves (high internalization) are presented in Fig. 3A. A three-dimensional reconstruction of fluorescent images taken at the same leaf region further demonstrates the internal localization of the
GFP-tagged bacteria (Fig. 3B). In contrast to arugula, essentially no internalization occurred in parsley, and fluorescent Salmonella cells were only seen on the leaf surface (Fig. 4A and B). To explore the notion that anatomical differences may account for the lower internalization efficiency in some of the leaves, scanning electron microscopy was used to visualize the fine details of the stomata and their surrounding region (Fig. 5). Stomatal images were taken from leaves displaying high (arugula), medium (basil), and low (parsley) internalization efficiencies. Representing images show multiple bacteria localized near and on the rim of a stomata with no apparent structural differences among the leaves (Fig. 5). The majority of stomata in all the
Fig. 4. Photomicrographs showing depth distribution of Salmonella cells in parsley leaf following internalization assay. (A) GFP-labeled bacteria are observed only on the leaf surface. Red fluorescence indicates auto-fluorescence of chlorophyll within chloroplasts. (B) A three-dimensional reconstruction of confocal microscopy images taken at the same leaf section shown above demonstrates the existence of Salmonella cells (green) on the epidermis only.
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
255
Fig. 5. Scanning electron microscopy images showing the topography of a single stomate region in the different leaves with multiple bacteria (apparently Salmonella) located around. Bar denotes 10 μm.
leaves remained partially opened following infection, implying that differences in stomatal closure were not responsible for the diverse behavior. 3.2. Internalization efficiency varies throughout the year The internalization efficiency varied considerably throughout the year and between years (0–100%; Fig. 6). High internalization occurred mainly in the summer months of 2008–9 (Jul.–Sep.), with no or lower rate during the winter time (Dec. 2008, Feb. 2009, and Oct. 2009–Mar. 2010). Nevertheless, high incidence of internalization was also recorded on Jan. 2008 (winter). 4. Discussion
Incidence (%) of internal Salmonella
The ability of human pathogens to penetrate stomata and reside at sites inaccessible to sanitizers represents a potentially unique mode of contamination, which might lead to failure of standard washing and sanitation procedures to remove or inactivate pathogens on leafy vegetables. Microscopic evidences for the ability of various human pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis to enter into the leaf apoplast were previously reported (Jha et al., 2005; Plotnikova et al., 2000; Prithiviraj et al., 2005). Similarly, E. coli O157:H7 was observed in the leaf interior underneath stomata of iceberg lettuce (Seo and Frank, 1999; Takeuchi and Frank, 2000, 2001a,b). Recently, we have studied Salmonella internalization in iceberg lettuce and demonstrated that bacterial entry was dependent on motility and chemotaxis likely towards photosynthetic products (sugars) found in the apoplast underneath stomata (Kroupitski et al., 2009). Since, leaf internalization may potentially represent an impor-
tant mode of contamination, either in the field or during processing of fresh produce, we have examined in the present study the ability of Salmonella to internalize edible leaves of three different lettuce varieties, as well as leaves of fresh herbs, namely basil, parsley, arugula. In addition, internalization was studied in non-edible tomato leaves. Considerable internalization was observed in 5 of the 7 leaves, however, essentially no internalization was evident in the case of parsley and tomato leaves. Since, significant differences in the incidence of internalization were observed between closely-related lettuce cultivars, it is difficult to target any potential physiological, biochemical or morphological trait that might affect internalization. Unlike, iceberg lettuce (Kroupitski et al., 2009) and basil (this study), Salmonella displayed no specific attraction to stomata in arugula, parsley (Figs. 2–4), or tomato leaves (data not shown). Yet, the incidence of Salmonella in the apoplast of arugula and parsley differed considerably. In the case of arugula, it might be assumed that the pathogen is attracted similarly to both the cuticle and stomata and therefore multiple bacteria can be seen on various areas on the cuticle as well as underneath stomata. The lack of apparent internalization in the case of parsley might be explained by inhibition of bacterial motility and/or chemotaxis toward stomata, or by the lack of sufficient amount of chemoattractants in the sub-stomatal apoplast. Essential oils produced by basil are known for their antimicrobial activity against a range of bacteria including S. Typhimurium (Hammer et al., 1999). It is possible that the relatively lower incidence of internalization (46 ± 12%) in basil compared to iceberg lettuce and arugula was affected by these antibacterial agents. Bacterial colonization of the phyllosphere varies in different plants and is influenced by the surface properties of the leaf, including morphology, chemical constituents and metabolic activities (Beuchat, 2002; Heaton and Jones, 2008; Leveau, 2009; Lindow and Brandl, 2003;
100 80 60 40 20 0 Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr
2008
2009
2010
Fig. 6. Incidence of internal Salmonella in iceberg lettuce leaves purchased between July 2008 and April 2010. Data represent the mean ± SD of 4 independent experiments each in triplicate.
256
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257
Yadav et al., 2005). SEM analysis revealed no obvious link between leaf morphology and the incidence of internalization, pointing perhaps to variations in the chemical properties of the tested leaves. An important feature of the results was the considerable variation in Salmonella internalization (0–100%) throughout the year and between years. High incidence of internalization was usually found in the summer months (July–September) in both 2008 and 2009. However, comparable internalization was observed also during Nov. and Dec. 2008, while essentially no internalization was found during the same months in 2009 (Fig. 6). It might be speculated that seasonal variations in day-length, light intensity, temperature, and humidity have influenced the content of chemoattractants in the leaves' apoplast and consequently caused variation in the incidence of internalization. In support of this notion, a seasonal difference in the microbial concentrations on leaves of fresh herbs has been reported. For example, in the case of cilantro and parsley, the concentrations of indicator bacteria were significantly higher in the fall than they were in spring or winter, and higher in spring than winter (Ailes et al., 2008). Similarly, seasonal conditions were shown to be an important factor in determining the fate of food-borne pathogens on fresh produce. Salmonella was detected at harvest on radish plants grown in contaminated field under conditions simulating wet and warm “summer” environment, but not under those simulating a comparatively drier and cooler “spring” environment (Natvig et al., 2002). Furthermore, competitiveness of Salmonella Thompson on wet cilantro leaves was significantly increased at plant incubation temperatures of 30 °C and 37 °C, enabling it to achieve higher population sizes than at 24 °C (Brandl and Mandrell, 2002). Although, higher internalization efficiencies were commonly recorded through summer months, a comparable high internalization incidence was also recorded once during a winter month (Jan. 2009). These findings, together with the month to month and year to year variations, suggest that other explanations, besides seasonal and climatic conditions might account for the different results. Since our study was carried on leafy vegetables that were purchased from a local supermarket; the identity of the iceberg lettuce variety is not known. It is possible that variation was simply related to differences in the lettuce cultivars used by farmers and sold in the supermarkets throughout the survey. A recent study, demonstrating different colonization efficiency in three spinach cultivars with different leaf surface morphologies (Mitra et al., 2009), further supports the importance of plant's cultivar in determining pathogen–plant interactions. Additional studies are required to clarify the relative role of seasonal parameters and the nature of the cultivar in the internalization process. 5. Conclusions Salmonella internalization seems to be variable among the seven different vegetables and fresh herbs examined in the present study; Two out of the seven (iceberg lettuce and arugula) displayed high incidence of internalization, 3 low to moderate (romaine and red lettuce, and basil, respectively), and 2 displayed very low incidence (parsley and tomato). Nonetheless, internalization of Salmonella in iceberg lettuce varied greatly (0–100%) from month to month and from year to year. Since internal Salmonella cells evade surface disinfection, it is highly important to assess leaf internalization in various leafy vegetables and fresh herbs. Identification of highly susceptible plants, as well as elucidation of plant- and environmentalfactors that affect internalization would be beneficial for the fresh produce sector and serve to better target its food safety resources. Acknowledgments This research was supported by Research Grant Award No. # US3949-06 from BARD, The United States - Israel Binational Agricultural
Research and Development Fund and by The Israeli Ministry of Agriculture, Chief scientist funds. English revision was done by Harriet Coleman.
References Ailes, E.C., Leon, J.S., Jaykus, L.A., Johnston, L.M., Clayton, H.A., Blanding, S., Kleinbaum, D.G., Backer, L.C., Moe, C.L., 2008. Microbial concentrations on fresh produce are affected by postharvest processing, importation, and season. Journal of Food Protection 71, 2389–2397. Beuchat, L.R., 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection 4, 413–423. Beuchat, L.R., Ryu, J.H., 1997. Produce handling and processing practices. Emerging Infectious Diseases 3, 459–465. Brandl, M.T., 2006. Fitness of human enteric pathogens on plants and implications for food safety. Annual Review of Phytopathology 44, 367–392. Brandl, M.T., Mandrell, R.E., 2002. Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere. Applied and Environmental Microbiology 68, 3614–3621. Franz, E., van Bruggen, A.H., 2008. Ecology of E. coli O157:H7 and Salmonella enterica in the primary vegetable production chain. Critical Reviews in Microbiology 34, 143–161. Hammer, K.A., Carson, C.F., Riley, T.V., 1999. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology 86, 985–990. Hanning, I.B., Nutt, J.D., Ricke, S.C., 2009. Salmonellosis outbreaks in the United States due to fresh produce: sources and potential intervention measures. Foodborne Pathogen Diseases 6, 635–648. Heaton, J.C., Jones, K., 2008. Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review. Journal of Applied Microbiology 104, 613–626. Horby, P.W., O'Brien, S.J., Adak, G.K., Graham, C., Hawker, J.I., Hunter, P., Lane, C., Lawson, A.J., Mitchell, R.T., Reacher, M.H., Threlfall, E.J., Ward, L.R., 2003. A national outbreak of multi-resistant Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 associated with consumption of lettuce. Epidemiology and Infection 130, 169–178. Itoh, Y., Sugita-Konishi, Y., Kasuga, F., Iwaki, M., Hara-Kudo, Y., Saito, N., Noguchi, Y., Konuma, H., Kumagai, S., 1998. Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts. Applied and Environmental Microbiology 64, 1532–1535. Jha, A.K., Bais, H.P., Vivanco, J.M., 2005. Enterococcus faecalis mammalian virulencerelated factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model. Infection and Immunity 73, 464–475. Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D., Sela, S., 2009. Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata. Applied and Environmental Microbiology 75, 6076–6086. Leveau, J.H.J., 2009. Microbiology: life on leaves. Nature 461, 741. Lindow, S.E., Brandl, M.T., 2003. Microbiology of the phyllosphere. Applied and Environmental Microbiology 69, 1875–1883. Mitra, R., Cuesta-Alonso, E., Wayadande, A., Talley, J., Gilliland, S., Fletcher, J., 2009. Effect of route of introduction and host cultivar on the colonization, internalization, and movement of the human pathogen Escherichia coli O157:H7 in spinach. Journal of Food Protection 72, 1521–1530. Natvig, E.E., Ingham, S.C., Ingham, B.H., Cooperband, L.R., Roper, T.R., 2002. Salmonella enterica serovar Typhimurium and Escherichia coli contamination of root and leaf vegetables grown in soils with incorporated bovine manure. Applied and Environmental Microbiology 68, 2737–2744. Nygard, K., Lassen, J., Vold, L., Andersson, Y., Fisher, I., Lofdahl, S., Threlfall, J., Luzzi, I., Peters, T., Hampton, M., Torpdahl, M., Kapperud, G., Aavitsland, P., 2008. Outbreak of Salmonella Thompson infections linked to imported rucola lettuce. Foodborne Pathogens and Disease 5, 165–173. Pakalniskiene, J., Falkenhorst, G., Lisby, M., Madsen, S.B., Olsen, K.E., Nielsen, E.M., Mygh, A., Boel, J., Molbak, K., 2009. A foodborne outbreak of enterotoxigenic E. coli and Salmonella Anatum infection after a high-school dinner in Denmark, November 2006. Epidemiology and Infection 137, 396–401. Pezzoli, L., Elson, R., Little, C., Fisher, I., Yip, H., Peters, T., Hampton, M., De Pinna, E., Coia, J.E., Mather, H.A., Brown, D.J., Nielsen, E.M., Ethelberg, S., Heck, M., de Jager, C., Threlfall, J., 2007. International outbreak of Salmonella Senftenberg in 2007. Eurosurveillance 12, E070614.3. Plotnikova, J.M., Rahme, L.G., Ausubel, F.M., 2000. Pathogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis. Plant Physiology 124, 1766–1774. Prithiviraj, B., Bais, H.P., Jha, A.K., Vivanco, J.M., 2005. Staphylococcus aureus pathogenicity on Arabidopsis thaliana is mediated either by a direct effect of salicylic acid on the pathogen or by SA-dependent, NPR1-independent host responses. The Plant Journal 42, 417–432. Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Martín-Belloso, O., 2009. Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. Comprehensive Reviews in Food Science and Food Safety 8, 157–180. Seo, K.H., Frank, J.F., 1999. Attachment of Escherichia coli O157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy. Journal of Food Protection 62, 3–9. Sivapalasingam, S., Friedman, C.R., Cohen, L., Tauxe, R.V., 2004. Fresh produce: growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. Journal of Food Protection 67, 2342–2353.
D. Golberg et al. / International Journal of Food Microbiology 145 (2011) 250–257 Takeuchi, K., Frank, J.F., 2000. Penetration of Escherichia coli O157: H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. Journal of Food Protection 63, 434–440. Takeuchi, K., Frank, J.F., 2001a. Confocal microscopy and microbial viability detection for food research. Journal of Food Protection 64, 2088–2102. Takeuchi, K., Frank, J.F., 2001b. Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection. Journal of Food Protection 64, 147–151. Tauxe, R., Kruse, H., Hedberg, C., Potter, M., Madden, J., Wachsmuth, K., 1997. Microbial hazards and emerging issues associated with produce; a preliminary report to the
257
national advisory committee on microbiologic criteria for foods. Journal of Food Protection 60, 1400–1408. Xicohtencatl-Cortes, J., Sánchez Chacón, E., Saldaña, Z., Freer, E., Girón, J.A., 2009. Interaction of Escherichia coli O157:H7 with leafy green produce. Journal of Food Protection 72, 1531–1537. Yadav, R.K.P., Karamanoli, K., Vokou, D., 2005. Bacterial colonization of the phyllosphere of Mediterranean perennial species as influenced by leaf structure and chemical features. Microbial Ecology 50, 185–196.