Size Matters: Biological and Food Safety Relevance of Leaf Damage for Colonization of Escherichia coli O157:H7 gfp

Abstract

This study examined the biological and food safety relevance of leaf lesions for potential invasion of food pathogens into the plant tissue (internalization). This was done by determining the role of artificial leaf damage in terms of damaged leaf area on proliferation of E. coli O157:H7 gfp+. In a two-factorial experiment, unwashed fresh baby leaf spinach (Spinacia oleracea L.) was subjected to four damage levels (undamaged, low, moderate, high damage; factor 1) and three incubation intervals (0, 1, 2 days post-inoculation; factor 2). Individual leaves were immersed for 15 s in a suspension loaded with E. coli O157:H7 gfp+ (10⁶ CFU × mL^-1). The leaves were analyzed individually using image analysis tools to quantify leaf area and number and size of lesions, and using confocal laser scanning and scanning electron microscopy to visualize leaf lesions and presence of the introduced E. coli strain on and within the leaf tissue. Prevalence of E. coli O157:H7 gfp+ was assessed using a culture-dependent technique. The results showed that size of individual lesions and damaged leaf area affected depth of invasion into plant tissue, dispersal to adjacent areas, and number of culturable E. coli O157:H7 gfp+ directly after inoculation. Differences in numbers of the inoculant retrieved from leaf macerate evened out from 2 days post-inoculation, indicating rapid proliferation during the first day post-inoculation. Leaf weight was a crucial factor, as lighter spinach leaves (most likely younger leaves) were more prone to harbor E. coli O157:H7 gfp+, irrespective of damage level. At the high inoculum density used, the risk of consumers' infection was almost 100%, irrespective of incubation duration or damage level. Even macroscopically intact leaves showed a high risk for infection. These results suggest that the risk to consumers is correlated with how early in the food chain the leaves are contaminated, and the degree of leaf damage. These findings should be taken into account in different steps of leafy green processing. Further attention should be paid to the fate of viable, but non-culturable, shiga-toxigenic E. coli on and in ready-to-eat leafy vegetables.

Keywords: enterohemorrhagic E. coli; food safety; internalization; leafy vegetables; lesions; risk assessment; shiga-toxigenic E. coli; spinach (Spinacia oleracea L.).

FIGURE 1

(A) Images of artificially damaged and Trypan blue-stained spinach leaves, visualizing the different levels of standardized artificial damage (from an undamaged leaf on the left to a high-damage leaf on the right). This batch of artificially damaged leaves was used for predictions of damage and number of lesions on E. coli O157:H7 inoculated leaves. (B) Plot of leaf weight (g) against leaf area (cm²), with regression line (red) showing the result of the model: y_{Leaf area} = β₀+β₁x_{Leaf weight}. (C) Predicted damage per leaf (%) on E. coli O157:H7 inoculated leaves (Wilcoxon test; ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (D) Predicted number of leaf lesions plotted against damaged leaf area (mm²) for different damage levels, where point size in scatter plots denotes leaf area of E. coli O157:H7 inoculated leaves.

FIGURE 2

Study conditions and potential processes following inoculation of E. coli O157:H7 gfp+ on leaves with various damage levels (undamaged, U; low damage, L; moderate damage, M; high damage, H). Leaf injury (bruising) modifies both leaf landscape (removal of abiotic barriers, e.g., cuticle; 3D-extension of surface area to colonize, see also Figure 5, Supplementary Figures 5–7, and Supplementary Videos 1, 2) and the pool of readily available nutrients. The nutrient pulse exhibited through bruising (M) alleviates biotic competition for space and nutrients, allowing E. coli to maintain a well-functioning metabolism, to substantially grow (dark green cell shade), and to disperse. In comparison, leaf landscape and resource pool alterations are less pronounced in low- and medium-damaged leaves, leaving E. coli to cope to with varying degrees of abiotic and biotic hurdles, leading to reduced growth rates due to depleted nutrient flux (medium green cells). Resource availability in damaged leaves varies over time. Wounds may not be viewed as a static culture. The nutrient pulse due to high damage is most pronounced directly after wounding, leading to resource utilization and concomitant decreases in growth rates during days after wounding. Under low and moderate damage, cells may be excited by a secondary nutrient pulse due to decaying leaf tissue. This, together with resuscitation of metabolically compromised cells, allows high growth rates. Thus, growth response of E. coli to leaf damage is dependent on the resource phase. Nutrient scarcity (U, L) leads to cell death (open cells with dark green frame) or loss of culturability (viable but not culturable, VBNC; light green cells with dark green frame). Cells may resuscitate from the latter when nutrient availability increases. Thus, reluctant E. coli growth is expected under such conditions (Illustration: B. Alsanius; SEM photos: E. Mulaosmanovic).

FIGURE 3

Relative distribution of E. coli O157:H7 gfp+ in suspensions after centrifugation (detachment, washing), after surface decontamination (leaf print), and in macerate of non-washed spinach leaves from a commercial leafy vegetable processing plant exposed to three levels of artificial damage (low, moderate, high) and leaves without intentional damage. Leaf analyses were performed directly after inoculation (0), and 1 and 2 days post-inoculation (dpi) of the target strain (n = 15).

FIGURE 4

(A) Log CFU E. coli O157:H7 gfp+ × cm^{– 2} leaf area in leaf macerate as a function of predicted leaf damage (%). Circle size in scatter plots denotes the leaf area of E. coli O157:H7 gfp+ inoculated leaves. (B) Boxplots of log CFU E. coli O157:H7 gfp+ found in leaf macerate after 0, 1, and 2 dpi, calculated per cm² of leaf area. Different letters above boxes indicate significant differences between damage levels (Tukey, p ≤ 0.05).

FIGURE 5

Bacterial colonization of artificially damaged spinach (Spinacia oleracea L.). Whole undamaged leaves (A–C), and leaves with a low (D–F), moderate (G–I), and high (J–L) level of artificial damage were dip-inoculated with 10⁶ CFU × mL^{– 1} E. coli O157:H7 bacteria and observed 2 dpi. Leaves were surface-sanitized with 0.25% sodium hypochlorite for 20 s before sampling for confocal laser scanning microscopy (CLSM). From scanning electron micrographs (SEM) (A,B,D,E,G,H,J,K) and CLSM images (C,F,I,L), it can be observed that lesion edges, junctions between adjacent epidermal cells, and stomata were the preferred colonization sites (arrows). Red fluorescence (C,F,I,L) indicates autofluorescence of the chlorophyll within chloroplasts, and green fluorescence indicates cells of E. coli O157:H7 bacteria. Lesions are marked with (Y). Scale bars 100 μm (A,D,G,J), 30 μm (B,E,H,K), and 20 μm (C,F,I,L).