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Distribution of Salmonella typhimurium in romaine lettuce leaves
Food Microbiology 28 (2011) 990e997
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Distribution of Salmonella typhimurium in romaine lettuce leaves Yulia Kroupitski a, Riky Pinto a, Eduard Belausov b, Shlomo Sela a, * a
Microbial Food-Safety Research Unit, Department of Food Quality and Safety, Institute for Postharvest Technology 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, Beth-Dagan, Israel
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2010 Received in revised form 16 January 2011 Accepted 25 January 2011 Available online 1 February 2011
Leafy greens are occasionally involved in outbreaks of enteric pathogens. In order to control the plant contamination it is necessary to understand the factors that influence enteric pathogeneplant interactions. Attachment of Salmonella enterica serovar typhimurium to lettuce leaves has been demonstrated before; however, only limited information is available regarding the localization and distribution of immigrant Salmonella on the leaf surface. To extend our knowledge regarding initial pathogeneleaf interactions, the distribution of green-fluorescent protein-labeled Salmonella typhimurium on artificially contaminated romaine lettuce leaves was analyzed. We demonstrate that attachment of Salmonella to different leaf regions is highly variable; yet a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log CFU g1) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU g1). Attachment to surfaces located at a central leaf region demonstrated intermediate attachment level (7.0 log CFU g1). Salmonella displayed higher affinity toward the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized underneath stomata within the parenchymal tissue, supporting the notion that this pathogen can also internalize romaine lettuce leaves. Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves, as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be correlated with a more composite leaf landscape. Our findings indicate that initial attachment of Salmonella to romaine lettuce leaf depends on multiple plant factors pertaining to the specific localization on the leaf tissue and to the developmental stage of the leaf. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Lettuce Attachment Internalization Stomata Leaf
1. Introduction Escherichia coli O157:H7 and various non-typhoidal Salmonella strains are among the most dominant bacterial agents linked to contamination of fresh and minimally-processed vegetables (Sivapalasingam et al., 2004; Hanning et al., 2009). Conventional washing and disinfection techniques apparently fail to completely eradicate microorganisms from fresh produce and only marginally reduce the number of pathogenic bacteria in experimentally contaminated vegetables (Zhuang et al., 1995; Beuchat, 1998; Seo and Frank, 1999; Raybaudi-Massilia et al., 2009).Therefore, in order to cope with the potential health hazards associated with consumption of contaminated products, it is vital to gain insight into the interactions between foodborne-pathogens and plants.
* Corresponding author. Tel.: þ972 3 9683750; fax: þ972 3 9683654. E-mail address: [email protected] (S. Sela). 0740-0020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.01.007
Vegetables and fruits differ in their surface morphology, tissue composition and metabolic activities and thus provide a wide range of diverse ecological niches (Beuchat, 2002; Heaton and Jones, 2008; Leveau, 2009). To gain insight into the colonization process of lettuce, Brandl and Amundson (2008) have recently examined the effect of leaf age on colonization of E. coli O157:H7 and Salmonella at pre- and post-harvest stages. Population size of both pathogens increased significantly on young (inner) leaves compared to middle leaves in the presence of free water on the leaves and warm temperature (28 C). Chemical analysis of the exudates collected from the surfaces of leaves of different ages showed that young-leaf exudates were richer in total nitrogen and carbon, suggesting that nitrogen might be the limiting growth factor in the older leaves (Brandl and Amundson, 2008). A number of other studies have investigated the interactions between enteric pathogens (Salmonella enterica and E. coli O157:H7) and leafy vegetables (Takeuchi et al., 2000; Takeuchi and Frank, 2001a; Brandl and Mandrell, 2002; Barak et al., 2007, 2008; Klerks et al., 2007; Shi et al., 2007; Franz and van Bruggen, 2008; Berger et al.,
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2009; Kroupitski et al., 2009a,b). The ability of human pathogens to colonize the phyllosphere depends greatly on their ability to attach to the leaf surface, (Brandl, 2006; Critzer and Doyle, 2010; Warriner and Namvar, 2010). During our studies on the attachment of S. enterica serovar typhimurium (S. typhimurium) to lettuce leaves, we observed that the distribution of the pathogen on the leaf surface is highly heterogeneous (Kroupitski et al., 2009a,b). This might have been related to the specific landscape topography or to biochemical properties immigrating bacteria encounter following introduction onto various regions on the leaf surface. In the present study we sought to examine in more details a potential relationship between the level of attachment of S. typhimurium and the location of the pathogen on lettuce leaves.
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Each attachment assay was performed in triplicate and repeated three times on different days with different lettuce. To quantify bacterial attachment to the adaxial and abaxial sides of the same leaf region, leaf discs were cut into smaller pieces (10 5 mm) following an attachment assay, and the occurrence of GFP-tagged Salmonella was determined by confocal laser-scanning microscopy, essentially as described before (Kroupitski et al., 2009a). The incidence of Salmonella on both surfaces was determined by scoring the number of microscopic fields (objective 40) harboring GFP-tagged bacteria at 5 attachment categories (0, 1e10, 10e50, 50e100, >100 cells per field) in 30 randomly chosen fields. The experiments were performed in triplicates and repeated twice at different days, with different lettuce. The data are presented as the average incidence (%) of attachment (SD).
2. Materials and methods 2.4. Scanning electron microscopy 2.1. Lettuce preparation Romaine lettuce heads were purchased from a local retail store and kept at 4 C for a maximum of 2 days before the onset of the experiments. The outermost leaves of the lettuce head were aseptically removed and the next 2 layers of leaves were detached and used for the experiments. Leaf discs (2.3 cm in diameter) were cut using an aseptic cork borer. For evaluation of attachment to different leaf areas, discs were prepared from three sites, one close to the stem, one from a central region and one from the far-end of the leaf blade (Fig. 1A). For evaluation of the attachment to leaves of different ages, the lettuce heads were separated into three age groups, old (two outermost layers), middle (twoethree inner leaves), and young (three innermost developed leaves) and discs were prepared from a central region of each leaf as described above. 2.2. Bacterial strain and inoculum preparation Salmonella typhimurium SL1344 strain expressing a green-fluorescent protein (GFP) was used in this study (Kroupitski et al., 2009b). Bacterial cultures were kept in LuriaeBertani (LB; 10 g Bacto-peptone, 5 g Yeast Extract, 10 g NaCl) broth containing 25% glycerol at 20 C. For each experiment, a fresh culture was made by growing Salmonella in LB broth supplemented with streptomycin (100 mg ml1) and nalidixic acid (30 mg ml1) for 18e20 h at 37 C. Bacteria were washed twice with sterile deionized water (SDW) by centrifugation at 2700g for 10 min. Inocula were made by resuspending the pellet in 10 ml SDW to a final concentration of 8 log10 colony-forming units (CFU) per ml. 2.3. Attachment assays Five lettuce discs derived from different leaves were submerged in 10 ml of GFP-tagged Salmonella suspension in SDW, containing 8 log10 CFU ml1 in a 50 ml sterile polypropylene tube (Labcon, Petaluma, CA) for 2 h at 25 C. Lettuce pieces were then rinsed twice for 1 min each in 50 ml SDW to remove unattached bacteria. The discs were transferred into 100 ml of 0.1% peptone water in sterile polyethylene bags (Interscience, St. Nom, France) and stomached for 2 min at maximal speed (Interscience BagMixer 400, St. Nom, France) to release leaf-associated Salmonella. The bacterial suspension was serially diluted in 0.1% peptone water and the number of colony-forming units was determined by plating on LB agar (Difco, Sparks, MD) containing streptomycin and nalidixic acid following incubation at 37 C for 24 h. Ten colonies from each plate were transferred onto xylose lysine deoxycholate (XLD) selective agar (Acumedia, Baltimore, MD, USA) plate to confirm the presence of Salmonella. Control experiments (without Salmonella) yielded no bacterial growth on LB plates supplemented with two antibiotics.
Lettuce leaf pieces 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 then washed again and internal 2 2 mm squares were excised, and processed for scanning electron microscopy as described before (Kroupitski et al., 2009b). Observation was performed under a Jeol JSM 35C-scanning electron microscope (Tokyo, Japan). 2.5. Stomatal density measurement Stomatal density was evaluated by confocal microscopy in both adaxial and abaxial sides of the same leaf piece by recording the number of stomata in 30 randomly chosen microscopic fields (40 objective). Stomatal density was calculated by dividing the average number of stomata per one microscopic field by the observed surface area. The experiment was repeated twice at different days, with different lettuce. 2.6. Statistical methods Statistical calculations were performed by ANOVA TukeyeKramer multiple Comparisons test using Instat version 3.0.6 (GraphPad Software, Inc., La Jolla, CA). 3. Results 3.1. Distribution of S. typhimurium on lettuce leaf In order to examine a potential relationship between leaf localization and the level of attachment, S. typhimurium attachment to various leaf blade regions was investigated (Fig. 1). Leaf discs were taken near the edge of the blade (region I), at the center (region II) and from a region near the petiole (region III). Maximal attachment was observed on surfaces localized near the petiole, and the level of attachment decreased towards the center (0.4 log CFU g1 reduction) and the far-end of the leaf blade (0.8 log CFU g1 reduction) (Fig. 1A). To corroborate the bacteriological findings, attachment was further studied by confocal microscopy (Fig. 1B). Indeed, a higher density of GFP-tagged bacteria was observed on leaf surfaces closer to the petiole (region III), supporting the viable count-based data (Fig. 1B). To investigate possible correlation between leaf surface topography and the level of attachment, leaf samples from the three tested locations were examined by scanning electron microscopy (SEM). Indeed, differences in the topography were visualized with the most complex topography found in surfaces localized near the petiole (Fig. 1B). Previous studies have reported the presence of a higher microbial population on vein regions (reviewed in Brandl, 2006). To
Fig. 1. Viable counts of S. typhimurium attachment to various leaf regions. Attachment of Salmonella romaine lettuce leaf (left panel) was tested by incubating the pathogen with leaf discs taken from three regions: (I) near the edge of the blade; (II) at the center of the leaf; (III) near the petiole. (A). The data represent the average log10 CFU g1 leaf tissue and standard deviation of three independent experiments with 3 replicates each. Small letters indicate significant difference (P < 0.05) between means according to ANOVA TukeyeKramer Multiple Comparisons Test.(B) Representative SEM and confocal microscopy micrographs showing surface topography and the distribution of GFP-labeled S. typhimurium (green), respectively at the three leaf regions. White arrows in panel II point to the presence of S. typhimurium in association with stomata. Red color represents autofluorescence of chloroplast. Bars on SEM and confocal microscopy images, denote 100 and 50 mm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article)
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a higher density of stomata (Beattie and Lindow, 1995). However, no significant difference in stomatal density (95 5.8 versus 95 10.8 stomata per 1 mm2) was recorded. 3.3. Comparative attachment of S. typhimurium to lettuce leaves of different ages In a recent study, Brandl and Amundson (2008) have reported increased colonization of young romaine lettuce leaves by Salmonella and E. coli O157:H7 compared to old leaves. To investigate if such trend might be related to variations in the initial attachment level, the attachment of Salmonella to young, intermediate and old leaves of the same lettuce head was quantified (Fig. 5A). Old leaves displayed higher attachment compared to young leaves with a significant decrease ranging from 0.69 to 1.56 log CFU g1 in intermediate and young leaves, respectively (P < 0.05). SEM analysis revealed that the level of attachment seems to be correlated to the landscape complexity of the tested surface (Fig. 5B), as observed above. 4. Discussion
Fig. 2. Photomicrograph of S. typhimurium near a vein region in romaine lettuce leaf. A representative confocal microscopy image taken from a central leaf region demonstrates the distribution of tagged (green) Salmonella cells (yellow arrows) on the leaf. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
determine if S. typhimurium attaches at higher affinity to these regions, the localization of the pathogen near veins was also investigated. Confocal microscopy observations of vein regions didn’t show any preference for attachment to such sites in contrast to other studies (Fig. 2). In another study, we have shown that S. typhimurium aggregates near and within stomata of iceberg lettuce leaf (Kroupitski et al., 2009b). We therefore examined whether Salmonella displays a similar behavior on romaine lettuce leaves. Confocal microscopy images showed no apparent attraction of GFP-tagged bacteria to stomata (Fig. 1B). Nevertheless, rarely, fluorescent bacteria were also found within stomata and at various depths below the leaf surface in the intercellular space (Fig. 3A,B). A three-dimensional reconstruction model of the fluorescent images taken at various depths of the same leaf region (Fig. 3C), further supports the finding regarding the internal localization of Salmonella. 3.2. Comparative attachment of S. typhimurium to the adaxial and abaxial sides of the leaf To examine potential difference in the attachment of Salmonella to surfaces on the abaxial and adaxial leaf sides, confocal microscopy images were taken from two sides of the same leaf region following bacterial attachment. Fluorescent Salmonella cells were more abundant on the abaxial as compared to the adaxial leaf side. For example, the incidence of Salmonella at the level of >100 bacteria per field was 40 21% at the abaxial surfaces compared to 0.5 1.4% at the adaxial side (Fig. 4A). Representative confocal microscopy images of the two surfaces are shown in Fig. 4B. To test whether the different attachment levels were related to topographical variations between the two surfaces, the landscape of the two leaf sides was examined by SEM. Indeed, the abaxial surface displayed much higher structural heterogeneity with complex topography compared to the adaxial surface (Fig. 4B). It was previously reported that the higher bacterial population numbers generally observed on the abaxial leaf surfaces, were related to
Attachment is the first step in the establishment of bacterial colonization on any surface, including the leaf surface. Attachment is mediated by a multiplicity of microbial and plant ligands and receptors, which jointly determine the attachment efficiency of bacteria to plant. Several studies have reported on the involvement of specific bacterial factors in the attachment of pathogenic E. coli and Salmonella to plant tissue (Barak et al., 2002, 2005, 2007; Jeter and Matthysse, 2005; Torres et al., 2005; Klerks et al., 2007; Berger et al., 2009; Teplitski et al., 2009; Warriner et al., 2009). Bacterial colonization of the phyllosphere depends upon a number of plant features, including leaf surface structure, chemical constituents present on the surface, as well as metabolic activities (Beattie and Lindow, 1995; Beuchat, 2002; Yadav et al., 2005; Brandl, 2006; Heaton and Jones, 2008). Although, attachment is considered to be a critical step in plant colonization by foodborne-pathogens, data on the initial attachment are still scarce. In the present study we aimed at finemapping the distribution of S. typhimurium on romaine lettuce leaves following initial attachment. Our results demonstrate a highly heterogeneous attachment pattern even in a given leaf. Nevertheless, we found a constant trend in the attachment pattern, which was related to the geography of the tested surface and its location on the abaxial or adaxial side of the leaf. Maximal attachment occurred at surfaces localized closer to the petiole and on the abaxial side. It was reported that plant pathogens also attach better to the abaxial side of the leaf and that a higher attachment may be in correlation with a higher stomatal density (reviewed in Beattie and Lindow, 1995). On the other hand, no significant correlation was found between epiphytic bacteria population size and stomatal density (Yadav et al., 2005). In the case of romaine lettuce, we observed no difference in the number of stomata at both sides of the same leaf, supporting the notion that morphological and perhaps other plant factors affect the attachment of S. typhimurium to romaine lettuce. Indeed, qualitative SEM analysis suggests a correlation between sites that support higher attachment level and leaf topography. These finding are in agreement with a recent study, which showed variation in E. coli O157:H7 colonization efficiency in three spinach cultivars differing in their leaf surface morphology (Mitra et al., 2009). Similarly, it was reported that in a perennial plant (Vaccinium macrocarpon), leaf surface topography was correlated with microbe colonization. Higher microbial populations were found on old leaves that displayed much rougher surfaces than those of young
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Fig. 3. Stomatal internalization of S. typhimurium in romaine lettuce. Confocal microscopy image showing Salmonella cells residing within a single stoma of lettuce leaf (A). Photomicrographs showing depth-distribution along a z-section of Salmonella cells in lettuce leaf tissue (B). Fluorescent images are overlaid with DIC images. Bacteria were observed within and beneath stomata (indicated by white arrows) in the intercellular space of the underlying parenchyma cells. Red fluorescence indicates autofluorescence of chlorophyll. A three-dimensional reconstruction of confocal microscopy images taken at the same leaf section shown above is also presented (C). The units of the X, Y, and Z axes are in micrometers. The yellow color corresponds to the localization of bacteria (green) and chloroplast (red) in close proximity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
leaves (Mechaber et al., 1996). The authors suggest that changes in leaf topography directly influence the ecology of the phyllosphere. Several other traits are considered to affect microbial population size on the phyllosphere. For example, in Mediterranean perennial plant species, positive correlation was observed between the number of epiphytic bacteria and the trichome density, while negative correlation was found between the size of the population and the thickness of the leaf, of the mesophyll, and of the abaxial epidermis (Yadav et al., 2005). Increased colonization of veins’ regions by plant epiphytic bacteria as well as by human pathogens was frequently noticed (Beattie and Lindow, 1995; Takeuchi and Frank, 2001b; Brandl and Mandrell, 2002; Lindow and Brandl, 2003; Brandl, 2006). It was suggested that high nutrient level and enhanced wettability of these sites might be responsible for the predominant attachment of microorganisms at these areas. Nevertheless, we didn’t observe such a trend with regard to S. typhimurium attachment. The different results may be due to variation in the experimental setting, i.e. the attachment assay, and the nature of plant and bacteria used. Escherichia coli O157:H7 and S. typhimurium were previously shown to attach preferentially to stomata of iceberg lettuce (Seo and Frank, 1999; Takeuchi and Frank, 2001a; Kroupitski et al., 2009b). This association frequently results in stomatal internalization by the human pathogens. We have shown that Salmonella internalization in iceberg lettuce involved chemotaxis and motility toward stomata (Kroupitski et al., 2009b). While, S. typhimurium cells were occasionally observed near stomata also in the case of
romaine lettuce, stomatal internalization was only rarely found. These findings might point to differences in the chemical compounds found around and within stomata in the two lettuce cultivars. Preliminary studies revealed no significant difference in sucrose content of post-harvested leaves between the two cultivars (S. Sela; data not shown). Therefore, further studies are needed to determine the factors that determine the distribution of Salmonella on lettuce leaves. Numerous studies have demonstrated an association between leaf age and population size of epiphytic microbial communities. For example, it was reported that epiphytic bacterial densities in rosette-type leafy vegetables, including lettuce, increases from the inner toward outer layers of leaves (Ercolani, 1976; Geeson, 1979; King et al., 1991; Morris and Lucotte, 1993; Jacques et al., 1995; Jacques, 1996; Kondo et al., 2006). A similar behavior was recently reported also for the human pathogens, E. coli O157:H7 and S. typhimurium DT104. These pathogens attach to inner iceberg lettuce leaves at 1e 2 log CFU g1 lower numbers compared to outer leaves (Kondo et al., 2006). Our results do conform to these reports and infer the existence of common mechanisms that determine microbial attachment to leaves of different ages also present in romaine lettuce. Interestingly, it has been reported that under permissive environmental conditions (moisture and temperature) faster bacterial growth of E. coli O157:H7 and S. typhimurium is achieved on young than on old romaine lettuce leaves, and that the enhanced bacterial multiplication was correlated with a higher nitrogen content in the leaves (Brandl and Amundson, 2008). Based on these findings the
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Fig. 4. Distribution of S. typhimurium on abaxial and adaxial sides of lettuce leaf. The distribution of Salmonella cells in thirty microscopic fields (x40) was examined and the average incidence (%) of cells at each cell density category was calculated (A). Each experiment was repeated three times with different lettuce. Different letters indicate significant difference (P < 0.05) between means according to ANOVA TukeyeKramer Multiple Comparisons Test.Representative SEM and confocal microscopy images demonstrating difference in the topography (SEM) and the number of GFP-tagged Salmonella (confocal) between the abaxial and adaxial leaf sides (B).
authors suggest that the young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7 (Brandl and Amundson, 2008). Our findings imply that a higher attachment doesn’t necessarily correlates with a higher multiplication rate, and infer that different plant factors are involved in the two processes. Contamination of mature lettuce might occur in the field, during harvest as well as during the industrial post-harvest handling. The industrial processing of ready-to-eat leafy vegetables is particularly prone to cross-contamination and deleterious outcome on the
safety of the final product due to massive produce quantities involved. Based on the studies regarding the attachment pattern in leaves of different ages, one might postulate that external leaves may pose a greater risk for contamination compared to young leaves, especially under conditions which don’t support bacterial multiplication. Indeed, removal of outermost leaves is a common procedure in the fresh produce industry prior to processing of lettuce heads. Further studies should elucidate at the molecular level the nature of bacterial and plant factors, which influence the initial
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Fig. 5. Attachment of S. typhimurium to romaine lettuce leaves of different ages. Viable Salmonella counts were enumerated on leaf discs taken from a central leaf region following attachment (A). The data represent the average log CFU g1 and standard deviation of three independent experiments. Different letters indicate significant difference (P < 0.05) between means according to ANOVA TukeyeKramer Multiple Comparisons Test. (B) Representative photographs of young (Y), middle (M) and old (O) lettuce leaves showing the topography (SEM) and the present of GFP-labeled S. typhimurium (confocal) on leaves of different ages (bacteria are indicated by yellow arrows). Images of leaf discs of comparable location to that employed in A are shown. Bars in SEM images and confocal microscopy images denote 100 and 50 mm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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attachment. Such knowledge might be helpful in devising novel anti-attachment control measures. Acknowledgments This study was supported by Research Grant Award no. # US3949-06 from BARD, The United StateseIsrael Binational Agricultural Research and Development Fund and The Israeli Ministry of Agriculture, Chief scientist funds. References Beattie, G.A., Lindow, S.E., 1995. The secret life of foliar bacterial pathogens on leaves. Annu. Rev. Phytopathol. 33, 145e172. Barak, J.D., Whitehand, L.C., Charkowski, A.O., 2002. Differences in attachment of Salmonella enterica serovars and Escherichia coli O157:H7 to alfalfa sprouts. Appl. Environ. Microbiol. 68, 4758e4763. Barak, J.D., Gorski, L., Naraghi-Arani, P., Charkowski, A.O., 2005. Salmonella enterica virulence genes are required for bacterial attachment to plant tissue. Appl. Environ. Microbiol. 71, 5685e5691. Barak, J.D., Jahn, C.E., Gibson, D.L., Charkowski, A.O., 2007. The role of cellulose and O-antigen capsule in the colonization of plants by Salmonella enterica. Mol. PlanteMicrobe Interact. 20, 1083e1091. Barak, J.D., Liang, A., Narm, K.E., 2008. Differential attachment to and subsequent contamination of agricultural crops by Salmonella enterica. Appl. Environ. Microbiol. 74, 5568e5570. Berger, C.N., Shaw, R.K., Brown, D.J., Mather, H., Clare, S., Dougan, G., et al., 2009. Interaction of Salmonella enterica with basil and other salad leaves. ISME J. 3, 261e265. Beuchat, L.R., 1998. Surface decontamination of fruits and vegetables eaten raw: a review. WHO/FSF/FOS/ 98.2. http://www.nutricion.org/publicaciones/pdf/ desinfeccion_hortalizas.pdf. Beuchat, L.R., 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes Infect. 4, 413e423. Brandl, M.T., 2006. Fitness of human enteric pathogens on plants and implications for food safety. Annu. Rev. Phytopathol. 44, 367e392. Brandl, M.T., Amundson, R., 2008. Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica. Appl. Environ. Microbiol. 74, 2298e2306. Brandl, M.T., Mandrell, R.E., 2002. Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere. Appl. Environ. Microbiol. 68, 3614e3621. Critzer, F.J., Doyle, M.P., 2010. Microbial ecology of foodborne pathogens associated with produce. Curr. Opin. Biotechnol. 21, 125e130. Ercolani, G.L., 1976. Bacteriological quality assessment of fresh marketed lettuce and fennel. Appl. Environ. Microbiol. 31, 847e852. Franz, E., van Bruggen, A.H., 2008. Ecology of E. coli O157:H7 and Salmonella enterica in the primary vegetable production chain. Crit. Rev. Microbiol. 34, 143e161. Geeson, J.D., 1979. The fungal and bacterial flora of stored white cabbage. J. Appl. Bacteriol. 46, 189e193. 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 Pathog. Dis 6, 635e648. Heaton, J.C., Jones, K., 2008. Microbial contamination of fruit and vegetables and the behaviour of enteropathogensin the phyllosphere: a review. J. Appl. Microbiol. 104, 613e626. Jacques, M.A., Kinkel, L.L., Morris, C.E., 1995. Population sizes, immigration and growth of epiphytic bacteria on leaves of different ages and positions of fieldgrowth endive (Cichorium endiva var. latifolia). Appl. Environ. Microbiol. 61, 899e906. Jacques, M.A., 1996. The effect of leaf age and position on the dynamics of microbial populations on aerial plant surfaces. In: Morris, C., Nicot, P., Nguyen-The, C. (Eds.), Aerial Plant Surface Microbiology. Plenum Press, New York, NY, pp. 233e248. Jeter, C., Matthysse, A.G., 2005. Characterization of the binding of diarrheagenic strains of E. coli to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts. Mol. PlanteMicrobe Interact. 18, 1235e1242.
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King, A.D., Magnuson, J.A., Torok, T., Goodman, N., 1991. Microbial flora and storage quality of partially processed lettuce. J. Food Sci. 56, 459e461. Klerks, M.M., Franz, E., van Gent-Pelzer, M., Zijlstra, C., van Bruggen, A.H., 2007. Differential interaction of Salmonella enterica serovars with lettuce cultivars and plantemicrobe factors influencing the colonization efficiency. ISME J. 1, 620e631. Kondo, N., Murata, M., Isshiki, K., 2006. Efficiency of sodium hypochlorite, fumaric acid, and mild heat in killing native microflora and Escherichia coli O157:H7, Salmonella typhimurium DT104, and Staphylococcus aureus attached to fresh-cut lettuce. J. Food Prot. 69, 323e329. Kroupitski, Y., Pinto, R., Brandl, M.T., Belausov, E., Sela, S., 2009a. Interactions of Salmonella enterica with lettuce leaves. J. Appl. Microbiol. 106, 1876e1885. Kroupitski, Y., Golberg, D., Belausov, E., Pinto, R., Swartzberg, D., Granot, D., et al., 2009b. Internalization of Salmonella in leaves is induced by light and involves chemotaxis and penetration through open stomata. Appl. Environ. Microbiol. 75, 6076e6086. Leveau, J., 2009. Microbiology life on leaves. Nature 461, 741. Lindow, S.E., Brandl, M.T., 2003. Microbiology of the phyllosphere. Appl. Environ. Microbiol. 69, 1875e1883. Mechaber, W.L., Marshall, D.B., Mechaber, R.A., Jobe, R.T., Chew, F.S., 1996. Mapping leaf surface landscapes. Proc. Natl. Acad. Sci. U S A 93, 4600e4603. 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. J. Food Prot. 72, 1521e1530. Morris, C.E., Lucotte, T., 1993. Dynamics and variability of bacterial population density on leaves of field-grown endive destined for ready-to-use processing. Int. J. Food Sci. Technol. 28, 201e209. Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Soliva-Fortuny, R., Martín-Belloso, O., 2009. Control of pathogenic and spoilage microorganisms in fresh-cut fruits and fruit juices by traditional and alternative natural antimicrobials. CRFSFS 8, 157e180. 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. J. Food Prot. 62, 3e9. Shi, X., Namvar, A., Kostrzynska, M., Hora, R., Warriner, K., 2007. Persistence and growth of different Salmonella serovars on pre- and postharvest tomatoes. J. Food Prot. 70, 2725e3271. Sivapalasingam, S., Friedman, C.R., Cohen, L., Tauxe, R.V., 2004. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food Prot. 67, 2342e2353. Takeuchi, K., Matute, C.M., Hassan, A.N., Frank, J.F., 2000. Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Pseudomonas fluorescens to lettuce leaves. J. Food Prot. 63, 1433e1437. Takeuchi, K., Frank, J.F., 2001a. Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection. J. Food Prot. 64, 147e151. Takeuchi, K., Frank, J.F., 2001b. Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce. J. Food Prot. 64, 298e304. Teplitski, M., Barak, J.D., Schneider, K.R., 2009. Human enteric pathogens in produce: un-answered ecological questions with direct implications for food safety. Curr. Opin. Biotechnol. 20, 166e171. Torres, A.G., Jeter, C., Langley, W., Matthysse, A.G., 2005. Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures. Appl. Environ. Microbiol. 71, 8008e8015. Warriner, K., Huber, A., Namvar, A., Fan, W., Dunfield, K., 2009. Recent advances in the microbial safety of fresh fruits and vegetables. Adv. Food Nutr. Res. 57, 155e208. Warriner, K., Namvar, A., 2010. The tricks learnt by human enteric pathogens from phytopathogens to persist within the plant environment. Curr. Opin. Biotechnol. 21, 131e136. 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. Microb. Ecol. 50, 185e196. Zhuang, R.Y., Beuchat, L.R., Angulo, F.J., 1995. Fate of Salmonella montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine. Appl. Environ. Microbiol. 61, 2127e2131.
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Distribution of Salmonella typhimurium in romaine lettuce leaves Yulia Kroupitski a, Riky Pinto a, Eduard Belausov b, Shlomo Sela a, * a
Microbial Food-Safety Research Unit, Department of Food Quality and Safety, Institute for Postharvest Technology 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, Beth-Dagan, Israel
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2010 Received in revised form 16 January 2011 Accepted 25 January 2011 Available online 1 February 2011
Leafy greens are occasionally involved in outbreaks of enteric pathogens. In order to control the plant contamination it is necessary to understand the factors that influence enteric pathogeneplant interactions. Attachment of Salmonella enterica serovar typhimurium to lettuce leaves has been demonstrated before; however, only limited information is available regarding the localization and distribution of immigrant Salmonella on the leaf surface. To extend our knowledge regarding initial pathogeneleaf interactions, the distribution of green-fluorescent protein-labeled Salmonella typhimurium on artificially contaminated romaine lettuce leaves was analyzed. We demonstrate that attachment of Salmonella to different leaf regions is highly variable; yet a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log CFU g1) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU g1). Attachment to surfaces located at a central leaf region demonstrated intermediate attachment level (7.0 log CFU g1). Salmonella displayed higher affinity toward the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized underneath stomata within the parenchymal tissue, supporting the notion that this pathogen can also internalize romaine lettuce leaves. Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves, as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be correlated with a more composite leaf landscape. Our findings indicate that initial attachment of Salmonella to romaine lettuce leaf depends on multiple plant factors pertaining to the specific localization on the leaf tissue and to the developmental stage of the leaf. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Lettuce Attachment Internalization Stomata Leaf
1. Introduction Escherichia coli O157:H7 and various non-typhoidal Salmonella strains are among the most dominant bacterial agents linked to contamination of fresh and minimally-processed vegetables (Sivapalasingam et al., 2004; Hanning et al., 2009). Conventional washing and disinfection techniques apparently fail to completely eradicate microorganisms from fresh produce and only marginally reduce the number of pathogenic bacteria in experimentally contaminated vegetables (Zhuang et al., 1995; Beuchat, 1998; Seo and Frank, 1999; Raybaudi-Massilia et al., 2009).Therefore, in order to cope with the potential health hazards associated with consumption of contaminated products, it is vital to gain insight into the interactions between foodborne-pathogens and plants.
* Corresponding author. Tel.: þ972 3 9683750; fax: þ972 3 9683654. E-mail address: [email protected] (S. Sela). 0740-0020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.01.007
Vegetables and fruits differ in their surface morphology, tissue composition and metabolic activities and thus provide a wide range of diverse ecological niches (Beuchat, 2002; Heaton and Jones, 2008; Leveau, 2009). To gain insight into the colonization process of lettuce, Brandl and Amundson (2008) have recently examined the effect of leaf age on colonization of E. coli O157:H7 and Salmonella at pre- and post-harvest stages. Population size of both pathogens increased significantly on young (inner) leaves compared to middle leaves in the presence of free water on the leaves and warm temperature (28 C). Chemical analysis of the exudates collected from the surfaces of leaves of different ages showed that young-leaf exudates were richer in total nitrogen and carbon, suggesting that nitrogen might be the limiting growth factor in the older leaves (Brandl and Amundson, 2008). A number of other studies have investigated the interactions between enteric pathogens (Salmonella enterica and E. coli O157:H7) and leafy vegetables (Takeuchi et al., 2000; Takeuchi and Frank, 2001a; Brandl and Mandrell, 2002; Barak et al., 2007, 2008; Klerks et al., 2007; Shi et al., 2007; Franz and van Bruggen, 2008; Berger et al.,
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2009; Kroupitski et al., 2009a,b). The ability of human pathogens to colonize the phyllosphere depends greatly on their ability to attach to the leaf surface, (Brandl, 2006; Critzer and Doyle, 2010; Warriner and Namvar, 2010). During our studies on the attachment of S. enterica serovar typhimurium (S. typhimurium) to lettuce leaves, we observed that the distribution of the pathogen on the leaf surface is highly heterogeneous (Kroupitski et al., 2009a,b). This might have been related to the specific landscape topography or to biochemical properties immigrating bacteria encounter following introduction onto various regions on the leaf surface. In the present study we sought to examine in more details a potential relationship between the level of attachment of S. typhimurium and the location of the pathogen on lettuce leaves.
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Each attachment assay was performed in triplicate and repeated three times on different days with different lettuce. To quantify bacterial attachment to the adaxial and abaxial sides of the same leaf region, leaf discs were cut into smaller pieces (10 5 mm) following an attachment assay, and the occurrence of GFP-tagged Salmonella was determined by confocal laser-scanning microscopy, essentially as described before (Kroupitski et al., 2009a). The incidence of Salmonella on both surfaces was determined by scoring the number of microscopic fields (objective 40) harboring GFP-tagged bacteria at 5 attachment categories (0, 1e10, 10e50, 50e100, >100 cells per field) in 30 randomly chosen fields. The experiments were performed in triplicates and repeated twice at different days, with different lettuce. The data are presented as the average incidence (%) of attachment (SD).
2. Materials and methods 2.4. Scanning electron microscopy 2.1. Lettuce preparation Romaine lettuce heads were purchased from a local retail store and kept at 4 C for a maximum of 2 days before the onset of the experiments. The outermost leaves of the lettuce head were aseptically removed and the next 2 layers of leaves were detached and used for the experiments. Leaf discs (2.3 cm in diameter) were cut using an aseptic cork borer. For evaluation of attachment to different leaf areas, discs were prepared from three sites, one close to the stem, one from a central region and one from the far-end of the leaf blade (Fig. 1A). For evaluation of the attachment to leaves of different ages, the lettuce heads were separated into three age groups, old (two outermost layers), middle (twoethree inner leaves), and young (three innermost developed leaves) and discs were prepared from a central region of each leaf as described above. 2.2. Bacterial strain and inoculum preparation Salmonella typhimurium SL1344 strain expressing a green-fluorescent protein (GFP) was used in this study (Kroupitski et al., 2009b). Bacterial cultures were kept in LuriaeBertani (LB; 10 g Bacto-peptone, 5 g Yeast Extract, 10 g NaCl) broth containing 25% glycerol at 20 C. For each experiment, a fresh culture was made by growing Salmonella in LB broth supplemented with streptomycin (100 mg ml1) and nalidixic acid (30 mg ml1) for 18e20 h at 37 C. Bacteria were washed twice with sterile deionized water (SDW) by centrifugation at 2700g for 10 min. Inocula were made by resuspending the pellet in 10 ml SDW to a final concentration of 8 log10 colony-forming units (CFU) per ml. 2.3. Attachment assays Five lettuce discs derived from different leaves were submerged in 10 ml of GFP-tagged Salmonella suspension in SDW, containing 8 log10 CFU ml1 in a 50 ml sterile polypropylene tube (Labcon, Petaluma, CA) for 2 h at 25 C. Lettuce pieces were then rinsed twice for 1 min each in 50 ml SDW to remove unattached bacteria. The discs were transferred into 100 ml of 0.1% peptone water in sterile polyethylene bags (Interscience, St. Nom, France) and stomached for 2 min at maximal speed (Interscience BagMixer 400, St. Nom, France) to release leaf-associated Salmonella. The bacterial suspension was serially diluted in 0.1% peptone water and the number of colony-forming units was determined by plating on LB agar (Difco, Sparks, MD) containing streptomycin and nalidixic acid following incubation at 37 C for 24 h. Ten colonies from each plate were transferred onto xylose lysine deoxycholate (XLD) selective agar (Acumedia, Baltimore, MD, USA) plate to confirm the presence of Salmonella. Control experiments (without Salmonella) yielded no bacterial growth on LB plates supplemented with two antibiotics.
Lettuce leaf pieces 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 then washed again and internal 2 2 mm squares were excised, and processed for scanning electron microscopy as described before (Kroupitski et al., 2009b). Observation was performed under a Jeol JSM 35C-scanning electron microscope (Tokyo, Japan). 2.5. Stomatal density measurement Stomatal density was evaluated by confocal microscopy in both adaxial and abaxial sides of the same leaf piece by recording the number of stomata in 30 randomly chosen microscopic fields (40 objective). Stomatal density was calculated by dividing the average number of stomata per one microscopic field by the observed surface area. The experiment was repeated twice at different days, with different lettuce. 2.6. Statistical methods Statistical calculations were performed by ANOVA TukeyeKramer multiple Comparisons test using Instat version 3.0.6 (GraphPad Software, Inc., La Jolla, CA). 3. Results 3.1. Distribution of S. typhimurium on lettuce leaf In order to examine a potential relationship between leaf localization and the level of attachment, S. typhimurium attachment to various leaf blade regions was investigated (Fig. 1). Leaf discs were taken near the edge of the blade (region I), at the center (region II) and from a region near the petiole (region III). Maximal attachment was observed on surfaces localized near the petiole, and the level of attachment decreased towards the center (0.4 log CFU g1 reduction) and the far-end of the leaf blade (0.8 log CFU g1 reduction) (Fig. 1A). To corroborate the bacteriological findings, attachment was further studied by confocal microscopy (Fig. 1B). Indeed, a higher density of GFP-tagged bacteria was observed on leaf surfaces closer to the petiole (region III), supporting the viable count-based data (Fig. 1B). To investigate possible correlation between leaf surface topography and the level of attachment, leaf samples from the three tested locations were examined by scanning electron microscopy (SEM). Indeed, differences in the topography were visualized with the most complex topography found in surfaces localized near the petiole (Fig. 1B). Previous studies have reported the presence of a higher microbial population on vein regions (reviewed in Brandl, 2006). To
Fig. 1. Viable counts of S. typhimurium attachment to various leaf regions. Attachment of Salmonella romaine lettuce leaf (left panel) was tested by incubating the pathogen with leaf discs taken from three regions: (I) near the edge of the blade; (II) at the center of the leaf; (III) near the petiole. (A). The data represent the average log10 CFU g1 leaf tissue and standard deviation of three independent experiments with 3 replicates each. Small letters indicate significant difference (P < 0.05) between means according to ANOVA TukeyeKramer Multiple Comparisons Test.(B) Representative SEM and confocal microscopy micrographs showing surface topography and the distribution of GFP-labeled S. typhimurium (green), respectively at the three leaf regions. White arrows in panel II point to the presence of S. typhimurium in association with stomata. Red color represents autofluorescence of chloroplast. Bars on SEM and confocal microscopy images, denote 100 and 50 mm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article)
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a higher density of stomata (Beattie and Lindow, 1995). However, no significant difference in stomatal density (95 5.8 versus 95 10.8 stomata per 1 mm2) was recorded. 3.3. Comparative attachment of S. typhimurium to lettuce leaves of different ages In a recent study, Brandl and Amundson (2008) have reported increased colonization of young romaine lettuce leaves by Salmonella and E. coli O157:H7 compared to old leaves. To investigate if such trend might be related to variations in the initial attachment level, the attachment of Salmonella to young, intermediate and old leaves of the same lettuce head was quantified (Fig. 5A). Old leaves displayed higher attachment compared to young leaves with a significant decrease ranging from 0.69 to 1.56 log CFU g1 in intermediate and young leaves, respectively (P < 0.05). SEM analysis revealed that the level of attachment seems to be correlated to the landscape complexity of the tested surface (Fig. 5B), as observed above. 4. Discussion
Fig. 2. Photomicrograph of S. typhimurium near a vein region in romaine lettuce leaf. A representative confocal microscopy image taken from a central leaf region demonstrates the distribution of tagged (green) Salmonella cells (yellow arrows) on the leaf. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
determine if S. typhimurium attaches at higher affinity to these regions, the localization of the pathogen near veins was also investigated. Confocal microscopy observations of vein regions didn’t show any preference for attachment to such sites in contrast to other studies (Fig. 2). In another study, we have shown that S. typhimurium aggregates near and within stomata of iceberg lettuce leaf (Kroupitski et al., 2009b). We therefore examined whether Salmonella displays a similar behavior on romaine lettuce leaves. Confocal microscopy images showed no apparent attraction of GFP-tagged bacteria to stomata (Fig. 1B). Nevertheless, rarely, fluorescent bacteria were also found within stomata and at various depths below the leaf surface in the intercellular space (Fig. 3A,B). A three-dimensional reconstruction model of the fluorescent images taken at various depths of the same leaf region (Fig. 3C), further supports the finding regarding the internal localization of Salmonella. 3.2. Comparative attachment of S. typhimurium to the adaxial and abaxial sides of the leaf To examine potential difference in the attachment of Salmonella to surfaces on the abaxial and adaxial leaf sides, confocal microscopy images were taken from two sides of the same leaf region following bacterial attachment. Fluorescent Salmonella cells were more abundant on the abaxial as compared to the adaxial leaf side. For example, the incidence of Salmonella at the level of >100 bacteria per field was 40 21% at the abaxial surfaces compared to 0.5 1.4% at the adaxial side (Fig. 4A). Representative confocal microscopy images of the two surfaces are shown in Fig. 4B. To test whether the different attachment levels were related to topographical variations between the two surfaces, the landscape of the two leaf sides was examined by SEM. Indeed, the abaxial surface displayed much higher structural heterogeneity with complex topography compared to the adaxial surface (Fig. 4B). It was previously reported that the higher bacterial population numbers generally observed on the abaxial leaf surfaces, were related to
Attachment is the first step in the establishment of bacterial colonization on any surface, including the leaf surface. Attachment is mediated by a multiplicity of microbial and plant ligands and receptors, which jointly determine the attachment efficiency of bacteria to plant. Several studies have reported on the involvement of specific bacterial factors in the attachment of pathogenic E. coli and Salmonella to plant tissue (Barak et al., 2002, 2005, 2007; Jeter and Matthysse, 2005; Torres et al., 2005; Klerks et al., 2007; Berger et al., 2009; Teplitski et al., 2009; Warriner et al., 2009). Bacterial colonization of the phyllosphere depends upon a number of plant features, including leaf surface structure, chemical constituents present on the surface, as well as metabolic activities (Beattie and Lindow, 1995; Beuchat, 2002; Yadav et al., 2005; Brandl, 2006; Heaton and Jones, 2008). Although, attachment is considered to be a critical step in plant colonization by foodborne-pathogens, data on the initial attachment are still scarce. In the present study we aimed at finemapping the distribution of S. typhimurium on romaine lettuce leaves following initial attachment. Our results demonstrate a highly heterogeneous attachment pattern even in a given leaf. Nevertheless, we found a constant trend in the attachment pattern, which was related to the geography of the tested surface and its location on the abaxial or adaxial side of the leaf. Maximal attachment occurred at surfaces localized closer to the petiole and on the abaxial side. It was reported that plant pathogens also attach better to the abaxial side of the leaf and that a higher attachment may be in correlation with a higher stomatal density (reviewed in Beattie and Lindow, 1995). On the other hand, no significant correlation was found between epiphytic bacteria population size and stomatal density (Yadav et al., 2005). In the case of romaine lettuce, we observed no difference in the number of stomata at both sides of the same leaf, supporting the notion that morphological and perhaps other plant factors affect the attachment of S. typhimurium to romaine lettuce. Indeed, qualitative SEM analysis suggests a correlation between sites that support higher attachment level and leaf topography. These finding are in agreement with a recent study, which showed variation in E. coli O157:H7 colonization efficiency in three spinach cultivars differing in their leaf surface morphology (Mitra et al., 2009). Similarly, it was reported that in a perennial plant (Vaccinium macrocarpon), leaf surface topography was correlated with microbe colonization. Higher microbial populations were found on old leaves that displayed much rougher surfaces than those of young
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Fig. 3. Stomatal internalization of S. typhimurium in romaine lettuce. Confocal microscopy image showing Salmonella cells residing within a single stoma of lettuce leaf (A). Photomicrographs showing depth-distribution along a z-section of Salmonella cells in lettuce leaf tissue (B). Fluorescent images are overlaid with DIC images. Bacteria were observed within and beneath stomata (indicated by white arrows) in the intercellular space of the underlying parenchyma cells. Red fluorescence indicates autofluorescence of chlorophyll. A three-dimensional reconstruction of confocal microscopy images taken at the same leaf section shown above is also presented (C). The units of the X, Y, and Z axes are in micrometers. The yellow color corresponds to the localization of bacteria (green) and chloroplast (red) in close proximity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
leaves (Mechaber et al., 1996). The authors suggest that changes in leaf topography directly influence the ecology of the phyllosphere. Several other traits are considered to affect microbial population size on the phyllosphere. For example, in Mediterranean perennial plant species, positive correlation was observed between the number of epiphytic bacteria and the trichome density, while negative correlation was found between the size of the population and the thickness of the leaf, of the mesophyll, and of the abaxial epidermis (Yadav et al., 2005). Increased colonization of veins’ regions by plant epiphytic bacteria as well as by human pathogens was frequently noticed (Beattie and Lindow, 1995; Takeuchi and Frank, 2001b; Brandl and Mandrell, 2002; Lindow and Brandl, 2003; Brandl, 2006). It was suggested that high nutrient level and enhanced wettability of these sites might be responsible for the predominant attachment of microorganisms at these areas. Nevertheless, we didn’t observe such a trend with regard to S. typhimurium attachment. The different results may be due to variation in the experimental setting, i.e. the attachment assay, and the nature of plant and bacteria used. Escherichia coli O157:H7 and S. typhimurium were previously shown to attach preferentially to stomata of iceberg lettuce (Seo and Frank, 1999; Takeuchi and Frank, 2001a; Kroupitski et al., 2009b). This association frequently results in stomatal internalization by the human pathogens. We have shown that Salmonella internalization in iceberg lettuce involved chemotaxis and motility toward stomata (Kroupitski et al., 2009b). While, S. typhimurium cells were occasionally observed near stomata also in the case of
romaine lettuce, stomatal internalization was only rarely found. These findings might point to differences in the chemical compounds found around and within stomata in the two lettuce cultivars. Preliminary studies revealed no significant difference in sucrose content of post-harvested leaves between the two cultivars (S. Sela; data not shown). Therefore, further studies are needed to determine the factors that determine the distribution of Salmonella on lettuce leaves. Numerous studies have demonstrated an association between leaf age and population size of epiphytic microbial communities. For example, it was reported that epiphytic bacterial densities in rosette-type leafy vegetables, including lettuce, increases from the inner toward outer layers of leaves (Ercolani, 1976; Geeson, 1979; King et al., 1991; Morris and Lucotte, 1993; Jacques et al., 1995; Jacques, 1996; Kondo et al., 2006). A similar behavior was recently reported also for the human pathogens, E. coli O157:H7 and S. typhimurium DT104. These pathogens attach to inner iceberg lettuce leaves at 1e 2 log CFU g1 lower numbers compared to outer leaves (Kondo et al., 2006). Our results do conform to these reports and infer the existence of common mechanisms that determine microbial attachment to leaves of different ages also present in romaine lettuce. Interestingly, it has been reported that under permissive environmental conditions (moisture and temperature) faster bacterial growth of E. coli O157:H7 and S. typhimurium is achieved on young than on old romaine lettuce leaves, and that the enhanced bacterial multiplication was correlated with a higher nitrogen content in the leaves (Brandl and Amundson, 2008). Based on these findings the
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Fig. 4. Distribution of S. typhimurium on abaxial and adaxial sides of lettuce leaf. The distribution of Salmonella cells in thirty microscopic fields (x40) was examined and the average incidence (%) of cells at each cell density category was calculated (A). Each experiment was repeated three times with different lettuce. Different letters indicate significant difference (P < 0.05) between means according to ANOVA TukeyeKramer Multiple Comparisons Test.Representative SEM and confocal microscopy images demonstrating difference in the topography (SEM) and the number of GFP-tagged Salmonella (confocal) between the abaxial and adaxial leaf sides (B).
authors suggest that the young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7 (Brandl and Amundson, 2008). Our findings imply that a higher attachment doesn’t necessarily correlates with a higher multiplication rate, and infer that different plant factors are involved in the two processes. Contamination of mature lettuce might occur in the field, during harvest as well as during the industrial post-harvest handling. The industrial processing of ready-to-eat leafy vegetables is particularly prone to cross-contamination and deleterious outcome on the
safety of the final product due to massive produce quantities involved. Based on the studies regarding the attachment pattern in leaves of different ages, one might postulate that external leaves may pose a greater risk for contamination compared to young leaves, especially under conditions which don’t support bacterial multiplication. Indeed, removal of outermost leaves is a common procedure in the fresh produce industry prior to processing of lettuce heads. Further studies should elucidate at the molecular level the nature of bacterial and plant factors, which influence the initial
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Fig. 5. Attachment of S. typhimurium to romaine lettuce leaves of different ages. Viable Salmonella counts were enumerated on leaf discs taken from a central leaf region following attachment (A). The data represent the average log CFU g1 and standard deviation of three independent experiments. Different letters indicate significant difference (P < 0.05) between means according to ANOVA TukeyeKramer Multiple Comparisons Test. (B) Representative photographs of young (Y), middle (M) and old (O) lettuce leaves showing the topography (SEM) and the present of GFP-labeled S. typhimurium (confocal) on leaves of different ages (bacteria are indicated by yellow arrows). Images of leaf discs of comparable location to that employed in A are shown. Bars in SEM images and confocal microscopy images denote 100 and 50 mm, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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