In the last decade there have been more than 15 foodborne outbreaks linked to contaminated lettuce or salad [10,130,131]. This provided the impetus for initiation of studies of attachment of human pathogens in both pre-and postharvest lettuce model systems [127,132-137]. Recent studies of E. coli O157:H7 (EcO157) on store-bought lettuce indicated that cells attached in a relatively short time period, and that not all cells could be removed by vigorous washing or treatment with chlorine (Table 2.3) [133,134,138]. EcO157, and other human pathogens and microbes, often are most concentrated at cut surfaces since there are vast nutrient resources released that can be metabolized by human pathogens. Cut lettuce leaves immersed in a suspension of a strain of EcO157 (up to 108 CFU/ml) were exposed to fluorescent anti-EcO157 antibody and observed by confocal microscopy. EcO157 attached predominantly to the cut edges of leaves; fewer cells attached to the intact cuticle of leaves, but were observed attached near stomates, on trichomes , and concentrated on vein areas of the leaf . Strains of EcO157, pseudomonas, salmonella, and L. monocytogenes (Lm) attached to different regions of cut lettuce leaves, indicating different and specific mechanisms of attachment for different species or strains . Recent studies of EcO157 and postharvest lettuce have addressed the general nature and force of the plant-EcO157 interactions by measuring the effect of surfactants and other treatments. More hydrophobic surfactants were the most effective in detaching EcO157 from the leaf cuticle, but cells at cut edges remained attached . Attachment of EcO157 to the lettuce leaf surface was 0.8 log10 higher after treatment with CaCl2; treatment with NaCl had no significant effect . Neither CaCl2 nor NaCl, however, had any significant effect on attachment to the cut edges.
Interestingly, it was reported that the medium in which EcO157 cells were grown affected attachment. Cells grown in tryptic soy broth were more hydrophilic, produced more CPS, and attached better to edges of lettuce (0.4 log10) and to the surface of both lettuce and apple (0.8 to 1.0 log10) than those grown in nutrient broth, suggesting that CPS may be involved directly in attachment . These studies suggest that EcO157 has different mechanisms for attaching to different regions of lettuce leaves, possibly involving hydrophobic interactions, surface carbohydrates (CPS/LPS), neutralization of ionic charge, or bridging of anionic moieties by divalent cations.
In sprout models of EcO157-lettuce attachment, strains of EcO157 implicated in produce outbreaks attached to lettuce roots approximately one log10 better than did two of five nonpathogenic E. coli strains, indicating variability in attachment among strains . Attachment of EcO157 strains was highest to seed coats and roots compared to the shoots.
GFP-labeled EcO157 was observed under a fluorescence stereomicro-scope to bind in aggregates to the grooves and edges of the seed coats, and to small root hairs of sprouted seedlings . High concentrations of EcO157 added to soil prior to growth of lettuce seedlings resulted in pathogen bound to all parts of the plant. Aggregates of EcO157 cells were observed on cotyledon and root tissue of lettuce seedlings grown for 5 days in spiked soil . These studies illustrate the potential for E. coli to colonize both pre- and postharvest lettuce.
In similar studies, an EcO157-GFP strain spiked in manure-contaminated soil (104 to 108 CFU/g) was monitored by confocal microscopy for presence on lettuce plants grown in the soil and then treated with chlorine and HgCl2 . EcO157-GFP remained attached to the edible portion of treated lettuce seedlings grown in soil spiked with the highest concentration of EcO157 (108 CFU/g). In addition, cells were observed as aggregates on three-day-old leaf surfaces, with some cells present 45 ^ below the outer leaf surface. There are multiple potential routes of entry for human pathogens in plants (lateral roots, stomates, pores, cuts, lesions, "invasion"), but whether specific mechanisms of attachment are involved during internalization on preharvest produce is not known (Figure 2.1 and Figure 2.2).
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