While all three plant species differ in their leaf structure and topography, the first two were shown to support significant levels of Salmonella internalization. In contrast, nearly no internalization was shown in tomato leaves by confocal laser microscopy , making these leaves an ideal control system for assessing potential misinterpretation when using surface sterilization and viable count.Green-fluorescent protein -labeled S. enterica serovar Typhimurium SL1334 strain was used throughout the study. Bacterial culture was prepared and stored in Lysogeny broth supplemented with glycerol at −70°C, as described . For each experiment, fresh culture was prepared by plating the bacteria on a new LB plate supplemented with 100mg/ml streptomycin and 10mg/ml gentamicin for 24h at 37°C. Two to three single colonies were as inoculated into LB broth devoid of NaCl and grown at 37°C with shaking for 18–20h. Cultures were washed twice with sterile saline by centrifugation at 2,700 g for 10min, and the final pellet was resuspended in sterile saline. Bacterial concentration was determined by plating × 10-fold serial dilutions on LB agar supplemented with the two antibiotics.Inoculation of leaves was performed, essentially as described before , except for the incubation temperature. Briefly, a single tomato leaflet, Arabidopsis leaf, or lettuce piece were each submerged in a single 50-ml sterile polypropylene tube containing 30-ml saline. The leaves were illuminated for 20min under a light intensity of 150-μE m−2 s−1 at room temperature, and then,lettuce vertical farming the saline was removed and replaced with a bacterial suspension containing ca. 108 SalmonellaCFU/ml saline. While this high inoculum does not represent real-life conditions, such high inocula were previously used to study Salmonella internalization in vivo and in vitro .

The incubation proceeded for 2h at 40°C, a temperature that increases stomatal openings in multiple species to facilitate Salmonella internalization. The leaf samples were washed twice by dipping in fresh sterile saline for 1min each time to remove unattached bacteria. Salmonella attachment to the leaf surface and internalization was analyzed by confocal microscopy and viable count, as described below. Each experiment included three leaves of the same plant, each in a single tube and the three plants species were processed on the same day.Surface disinfection was performed using one of the three disinfectants, 1% sodium hypochlorite , 1% silver nitrate , and 70% ethanol . Briefly, whole leaves of Arabidopsis and tomato or lettuce leaf samples were submerged in 20-ml disinfectant solution with gentle agitation for 7min. Leaf samples were taken out after 1, 3, 5, and 7min and washed extensively by dipping the leaves four times in 20-ml sterile double-distilled water to remove the residual disinfectant solution. In order to avoid interference by bacteria that may enter through the cut tissues, an internal leaf disks were excised from the three leaves using a sterile corkborer. The leaf disks were aseptically cut into two identical pieces with a sterile scalpel, one was taken for bacterial extraction and viable count, and the other was taken for confocal microscopy. A high-speed benchtop homogenizer Fast Prep®-24 was used for the homogenization of the leaf samples in 2-ml micro-tubes containing glass beads and 500μl of buffer peptone water . Homogenization conditions were 4,000 rpm for 40 s at room temperature. Homogenate samples and 10× serial dilutions were spread plated into XyloseLysine-Desoxycolate agar supplemented with streptomycin and gentamicin in order to enumerate internalized Salmonella cells that presumably survived the disinfection treatment.

Inoculated leaves suspended for up to 7min in SDDW without disinfection and then washed in fresh SDDW served as non-treated control to determine the initial number of leaf-associated bacteria. Salmonella counts of control and treated samples were converted to log CFU/cm2 .Human pathogens can colonize plants and persist on and sometimes within various plant’s tissues, and upon consumption may cause foodborne diseases . Accordingly, accurate determination of the localization of human pathogen on or within leaves is vital for basic science as well as for developing new strategies for preventing and intervening to address the problem of fresh produce contamination. The determination of bacterial internalization in a plant is a function of, among others, the method used to assess bacterial localization . Ultimately, surface sterilization should completely inactivate external bacteria while leaving internalized bacteria intact. Still, only a few studies have systematically validated the efficacy of surface sterilization to kill surface-attached bacteria. In one such study, 13 disinfection conditions/methods were compared for their effectiveness in killing GFP-tagged E. coli O157:H7 on lettuce leaf surfaces using leaf imprints on agar media. Dipping in 80% ethanol for 10 s followed by immersion in 0.1% HgCl2 for 10min was reported to be the most effective disinfection method for inactivating both E. coli and Salmonella strains . However, no confocal microscopy study corroborated the results. Many studies have adopted previously reported protocols to inactivate surface-attached bacteria, even when utilizing different plants and/or bacterial strains . Bacteria may vary in their intrinsic tolerance to disinfectants and may preferentially reside at unique leaf-specific microsites , which may facilitate the protection of the colonized bacteria against disinfection . Consequently, a disinfection protocol developed for inactivating a specific Salmonella strain on the leaves of a particular plant cultivar may not fit all. Evidently, when a partial inactivation is achieved, some surface residing bacteria may be mis-classified as internal bacteria, while truly internalized bacteria killed due to permeation of the disinfectant into the intact leaf tissue may be mistakenly regarded as surface-attached bacteria. The present study provides data from a systematic comparison of leaf internalization through stomata by a GFP-tagged Salmonella Typhimurium strain in the leaves of the three plant species using surface sterilization and plate-count technique.

The study did not compare leaf internalization among plants but rather the effect of the various disinfection protocols on leaf internalization in each plant species. We used three disinfectants , commonly applied, alone or in combination with others, for sterilizing plant surfaces . To simplify the comparison between the protocols, we used a single concentration of the disinfectants, each time, and compared the effect of the sterilization time on quantifying viable bacteria, apparently representing internalized Salmonella cells. In parallel to the viable counts measurements, we utilized confocal microscopy to accurately assess bacterial localization on or within the leaf tissue. We initially examined leaf internalization in iceberg lettuce leaves previously shown by confocal microscopy to support Salmonella internalization . Indeed,vertical grow shelf confocal microscopy confirmed a high incidence of internalization in non-disinfected leaves; however, surface disinfection with all three agents resulted in reducing fluorescence, suggesting that the disinfectants seemingly penetrated the leaf tissues to some degree and injured the cells . A substantial decrease in the number of apparent internalized bacteria was observed using the plate-count method following 1 to 7 min treatment . The determination of leaf internalization by surface disinfection and viable count showed wide variations in the number of apparently internalized bacteria in leaves of each plant species, depending upon the type of the disinfectant and the treatment duration. These differences are likely attributed to the increased killing of leaf-associated bacteria with time or to the transition of a portion of the Salmonella population to the viable but non-culturable state . The observation of fluorescent cells on the leaf surface does not provide a clear indication regarding the presence of disinfection-tolerant bacteria, since the Salmonella strain carried a stable GFP , which may continue to emit fluorescence in VBNC bacteria, as well as in dead cells with intact GFP. Further studies using methods that can discriminate between live and dead bacteria are needed to determine the physiological status of the treated fluorescent bacteria on the leaves’ surface. Still, the possible entry of Salmonella into a VBNC state in the plant environment may lead to underestimation of both attachment and internalization when using the viable count assay alone. Based on the confocal microscopy studies, Salmonella displays a lower incidence of leaf internalization in Arabidopsis than in lettuce . Likewise, the viable count method demonstrated lower numbers of viable bacteria during all treatment times . All three agents displayed comparable surface disinfection effectiveness; however, they varied significantly in the apparent internalization . A 7-min treatment with 1% NaHClO or 1% AgNO3 resulted in the complete loss of fluorescent cells inside the leaf, suggesting that they efficiently penetrated the leaves and injured the internalized bacteria. In a previous report, we were not able to show internalization of the same Salmonella strain in tomato leaves . Consequently, the assessment of tomato leaf internalization, side by side, by the two methodologies provided a unique opportunity to assess the suitability of the tested disinfection conditions inactivate bacteria in the leaf surface. Evaluation of Salmonella internalization by confocal microscopy, with no surface sterilization, confirmed our inability to demonstrate the internalization of Salmonella in these tomato leaves with the techniques used. Usage of 1% NaHClO for 1 to 7min resulted in different numbers of apparent internalized bacteria, ranging from 4 logs CFU/cm2 to 0, respectively. Parallel confocal microscopy analysis of the treated leaf samples confirmed the lack of detection of leaf internalization, suggesting that only 7-min treatment resulted in sufficient killing of external bacteria in this model system. The use of 70% ethanol as a sole disinfectant for up to 7min failed to inactivate all external bacteria, as determined by viable counts, thus mistakenly suggesting the internalization of about 3 log CFU/cm2 .

Treatment with 1% AgNO3 resulted in substantial inactivation of surface attached bacteria in 1 and 3min treatment, while treatment duration of 5 and 7min was sufficient to kill all external bacteria, hence providing results comparable to those obtained by confocal microscopy. These findings indicate that non-validated surface sterilization conditions may lead to misinterpretation of the actual number of internalized bacterial cells. Notably, the apparent lack of leaf internalization of the tested S. typhimurium strain in the tomato cultivar used in this study , as well as in S. lycopersicon cv. MP1, tested previously , calls for further research. It is particularly interesting to examine whether the two cultivars are naturally resistant to leaf internalization of other Salmonella serovars and strains under more natural tomato growing conditions. Elucidation of the mechanisms involved in the inhibition of leaf internalization might prove important for understanding human pathogen plant interactions and developing new mitigation strategies for Salmonella internalization. Surface disinfection by treatment with 1% AgNO3 was less effective in lettuce compared to tomato leaves. These differences are likely correlate to specific leaf features, such as surface morphology and/or physico-chemical properties known to impact leaf colonization . Previous studies have already noted that the attachment of bacteria to specific micro-environments on the leaf, such as cavities and crevices on the leaf surface, may favor the persistence of surface-attached bacteria following disinfection . Altogether, this is the first time a systematic study reported a comparison of three surface sterilization protocols in leaves of three plants, side by side, with a confocal microscopy study. While the selection of an optimal disinfection protocol for each of the three plants was beyond the scope of this study, we have demonstrated the dependency of the apparent bacterial internalization on the disinfection conditions and shown the impact of the quantification method on the extent of leaf internalization. It should be noted that entry of bacterial pathogens into the leaf tissue might occur through stomata, hydathodes, and injured tissues or by transport through the roots and stem . In the present study, we utilized specific in vitro inoculation and experimental conditions to compare the effect of three surface disinfection protocols on Salmonella internalization through stomata. The study was not designed to investigate other factors that might affect bacterial internalization nor the different mode of leaf internalization. Therefore, we suggest interpretation of our results with caution, especially when comparing to other studies that used different inoculation models and surface disinfection protocols. Whole leaves or leaflets were used for inoculation in the case of Arabidopsis and tomato, respectively; however, in the case of lettuce, square leaf pieces were used, which potentially may enable direct access of bacteria into the apoplast through the injured tissue. However, previous confocal microscopy observations showed a limited penetration of Salmonella through the cut tissues , which did not affect the internal leaf tissue used for bacterial enumeration.