Internalization of Salmonella enterica in Leaves Is Induced by Light and Involves Chemotaxis and Penetration through Open Stomata

Abstract

Outbreaks of salmonellosis related to consumption of fresh produce have raised interest in Salmonella-plant interactions leading to plant colonization. Incubation of gfp-tagged Salmonella enterica with iceberg lettuce leaves in the light resulted in aggregation of bacteria near open stomata and invasion into the inner leaf tissue. In contrast, incubation in the dark resulted in a scattered attachment pattern and very poor stomatal internalization. Forcing stomatal opening in the dark by fusicoccin had no significant effect on Salmonella internalization. These results imply that the pathogen is attracted to nutrients produced de novo by photosynthetically active cells. Indeed, mutations affecting Salmonella motility and chemotaxis significantly inhibited bacterial internalization. These findings suggest a mechanistic account for entry of Salmonella into the plant's apoplast and imply that either Salmonella antigens are not well recognized by the stoma-based innate immunity or that this pathogen has evolved means to evade it. Internalization of leaves may provide a partial explanation for the failure of sanitizers to efficiently eradicate food-borne pathogens in leafy greens.

Minimal processing of iceberg lettuce has no substantial influence on the survival, attachment and internalization of E. coli O157 and Salmonella

“…When studying the surface, high densities of bacteria were found, but both pathogens behaved similar and were never able to reach the parenchyma through the stomata, they were only found in the substomatal cavities. These results were expected as Kroupitski et al (2009a) showed already that internalization did not (often) occur in the dark or at low temperature for Salmonella. They reported internalization in around 10% of the observed fields both at low light (3 μE m −2 s −1 ) in combination with low storage temperature (4°C) and also when the lettuce was stored in the dark (at 30°F ig.…”

“…internalization. Kroupitski et al (2009a) showed that the presence of exogenous nutrients can inhibit internalization as there is no need for the pathogens to search for other nutrients (Kroupitski et al, 2009a). Furthermore, we cannot exclude that the stomata were closed in the bag given the low metabolic state of the lettuce at low temperatures.…”

“…The results of our study are limited to lettuce stored at appropriate cold storage temperatures (7°C). As Kroupitski et al (2009a) has shown that internalization occurs more frequently at higher temperatures, it should be very interesting for future research to study higher temperatures and longer storage periods corresponding with time and temperature abuses. Finally, our results of internalization through stomata showed a large variability between the different independent repeats.…”

“…Since Kroupitski et al (2009a) revealed that Salmonella is attracted to nutrients produced by photosynthetically active cells of cut lettuce, and bagged fresh-cut lettuces are exposed to light in food stores, storage conditions (light or dark) were taken into account as well, In addition, this effect was for the first time investigated on E. coli O157 and cut lettuce. The classical plate count technique as well as quantitative confocal laser scanning microscopy were used in the present study to evaluate and quantify the effect of high (necessary for monitoring with confocal laser microscopy) or moderate pathogen levels.…”

Interacciones entre Escherichia coli O157:H7 y Plantas Comestibles. ¿Se han Desarrollado Mecanismos de Internalización Bacteriana?

“…Several studies have shown that E. coli O157:H7 is able to enter by natural openings in the plant surface, such as the sub-stomatal cavities in leaves (Brandl, 2008;Erickson, 2012;Kroupitski et al, 2009). Once the bacteria are inside the plant, following closure of the guard cells, they are protected from most superficial sanitisers (Gomes et al, 2009).…”