Food safety and vegetables: contamination of baby-leaf salads by human pathogens

Baby leaves are leafy vegetables harvested at an early stage of growth (up to the eighth true leaf) and consumed as salads. They can be marketed as unprocessed products or, more commonly, subjected to minimal processing and sold in a convenient ready-to-eat form, which offers the advantage of ease of consumption. It is well known that vegetables may host human pathogens like Salmonella enterica and Escherichia coli, possibly leading to the occurrence of foodborne diseases. From this point of view, the consumption of salads may raise particular concerns as they are eaten raw and often without prior proper washing. This thesis presents the results of three experiments on the issue of foodborne diseases in baby-leaf salads, with the final objective to provide information useful for increasing the safety of these products. The first and the second experiments aimed to highlight possible differences in the susceptibility to human pathogens contamination between 30 accessions of baby leaves belonging to different species and/or varieties and to understand the relationship between the susceptibility and leaf traits. In the first experiment the 30 accessions were surface inoculated with a suspension of E. coli ATCC 35218 containing 1 × 107 cells/mL and the attachment was measured 1.5 h after inoculation. Significant differences in attachment were detected between the accessions. The three most susceptible accessions (romaine lettuce ‘Bionda degli Ortolani’, Swiss chard, and rocket) and the three least susceptible (wild rocket ‘Yeti’, wild lettuce, and lamb’s lettuce ‘Trophy F1′) were selected and characterized for leaf micro‐morphological traits (stomata density and size, surface roughness) and water content. Scanning electron microscopy was used to analyse the stomatal parameters. Roughness was measured by an innovative portable 3D digital microscope. No significant correlation between the attachment of E. coli on the leaves and stomatal parameters was detected, while the attachment was positively correlated with roughness and water content. E. coli population in surface‐inoculated leaves was also measured after a UV treatment, which was found to be less effective in reducing bacterial contamination in the rougher leaves. This result suggested that roughness offers UV protection to E. coli cells, further highlighting its impact on the microbiological safety of baby-leaf greens. In the second experiment the attachment of S. enterica on the leaves of the 30 accessions was evaluated after 5 min of inoculation, revealing differences in susceptibility to contamination by S. enterica between the baby-leaf salads. Wild lettuce (Lactuca serriola L.) and lamb’s lettuce ‘Trophy F1’ (Valerianella locusta [L.] Laterr.) showed the lowest level of contamination, while sorrel was the most contaminated. The differences in attachment found between the salads were then related to the following leaf traits: hydrophobicity, roughness, and epicuticular waxes. Attachment was correlated to hydrophobicity (measured as contact angle) and epicuticular waxes, but not to roughness, presumably due to the short incubation time. The most important wax components for attachment were alcohols and, in particular, the 3-D wax crystals of C26 alcohol, but fatty acids probably also had a role. Both these compounds increased hydrophobicity. The presence of thymol, whose antimicrobial properties are well known, was found in lamb’s lettuce. Finally, the third experiment was conducted to preliminary evaluate fluorescence microscopy as a mean to directly track E. coli proliferation in baby leaves. Cultivated lettuce (romaine lettuce 'Bionda degli Ortolani') and wild lettuce, which had previously shown a different susceptibility to E. coli contamination, were used for the experiment. Fluorescence microscope observations were performed at time 0 and 24 h after inoculation. The fluorescence area increased over the 24-hour incubation interval. Wild lettuce showed a smaller area of fluorescence than romaine (-56.1%), and a fluorescence intensity approximately 19.1% lower, confirming to be less susceptible to E. coli contamination in comparison with cultivated lettuce. The results obtained in this thesis can turn into practical implications and give rise to some recommendations for growers. The screening of a large number of baby leaves for the susceptibility to contamination by E. coli and S. enterica highlighted the most and the least risky in terms of food safety. This information can guide growers’ choice and be of interest for the ready-to-eat industry. Besides, the thesis provided findings on leaf traits associated with a decrease in proliferation of human pathogens in salads. Such traits could be considered in breeding programs with the goal of obtaining cultivars less prone to human pathogens. Finally, although preliminary, the results of the third experiment indicated a new approach for real-time observation and monitoring of bacterial proliferation on leaf surfaces, providing valuable information in the field of plant-bacteria interactions. In conclusion, this work offers insights for enhancing the food safety of leafy vegetables.