Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs

Abstract

Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥ 1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (P < 0.05). Internalization incidence in fresh basil was 46 ± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0–100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization.

Introduction

Enteric diseases linked to consumption of fresh produce have dramatically increased in the last several decades (Sivapalasingam et al., 2004, Tauxe et al., 1997). Fresh produce-associated outbreaks result in high economical losses to farmers, distributors and the food industry. Most outbreaks in the U.S. related to consumption of leafy greens were associated with Escherichia coli O157:H7, however, Salmonella enterica outbreaks related to this type of produce have also been reported (Hanning et al., 2009). S. enterica outbreaks linked to leafy greens were also reported in Europe (Raybaudi-Massilia et al., 2009) and included an outbreak of S. Senftenberg in the UK, Denmark and the Netherlands (Pezzoli et al., 2007), S. Thompson outbreak in Scandinavia and UK linked to consumption of rocket leaves (Nygard et al., 2008) and an S. Anatum infection associated with imported basil in Denmark (Pakalniskiene et al., 2009). These outbreaks underline the challenges to the agro-industrial sector as well as to the public health authorities.

Fresh produce can become contaminated by human pathogens throughout the food-chain. It is accepted that plants might become contaminated in the field through the use of contaminated irrigation water and the use of animal manure for fertilization purposes (Beuchat and Ryu, 1997, Brandl, 2006, Franz and van Bruggen, 2008, Horby et al., 2003). Fresh produce can also become contaminated during harvest and at post-harvest stages due to poor workers' hygiene, and low sanitation in the processing plant (Beuchat and Ryu, 1997, Brandl, 2006).

Studies on the interactions between E. coli O157:H7 and cut-lettuce leaves demonstrated efficient attachment of bacteria to the surface, trichomes, stomata, and cut edges (Seo and Frank, 1999, Takeuchi and Frank, 2000, Takeuchi and Frank, 2001a, Takeuchi and Frank, 2001b). E. coli O157:H7 cells were occasionally observed to be entrapped 20 to 100 μm below the surface in stomata and cut-edges of iceberg lettuce (Seo and Frank, 1999). E. coli O157:H7 was also shown to colonize the inner tissues and stomata of cotyledons of radish sprouts, developed from seeds experimentally contaminated with the bacterium (Itoh et al., 1998). Recently, we have shown that Salmonella Typhimurium is capable of penetrating the epidermis of iceberg lettuce leaves through open stomata in a process that involves flagellar motility and chemotaxis (Kroupitski et al., 2009). In a recent study, E. coli O157:H7 was also shown to gain entry into internal tissue of baby spinach leaves. The internalization process required intact flagella and type three secretion system (Xicohtencatl-Cortes et al., 2009). In contrast, Mitra et al. (2009) found no conclusive evidence for natural entry of this pathogen into the interior of spinach leaves.

Since internalized bacteria are refractory to disinfection (Seo and Frank, 1999), bacterial internalization potentially poses a safety hazard to consumers. In order to assess the widespread stomatal internalization in leafy greens other than iceberg lettuce, we have examined the internalization of S. enterica sv. Typhimurium (S. Typhimurium) among several leafy vegetables and herbs.