Interactions of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes plants cultivated in a gnotobiotic system
Introduction
It is estimated that 14% of all foodborne illness cases reported in North America can be linked directly to minimally processed fruit and vegetables (Bean et al., 1997, Mead et al., 1999). Virulent pathogens such as Escherichia coli O157:H7, Salmonella and Listeria monocytogenes have all been implicated in such outbreaks of foodborne illness (Beuchat, 1996). To give examples, Es. coli O157:H7 has been associated with alfalfa sprouts, apple juice, cabbage, celery, cilantro, coriander, cress sprouts and lettuce (Beuchat, 2002); Salmonella has been recovered from tomatoes, seed sprouts, cantaloupe, apple juice, and orange juice (Beuchat, 2002); and L. monocytogenes has been linked with a diverse range of produce types including bean sprouts, cabbage, chicory, cucumber, eggplant, lettuce, mushroom, potatoes, radish, salad vegetable and tomato (Beuchat, 1996, National Advisory Committee on Microbiological Criteria for Food, 1999, Beuchat, 2002).
Through various studies, it has been established that the survival and interaction of human pathogens with growing plants are greater than previously thought (Warriner et al., 2003a). It has been suggested that pathogens, such as Es. coli O157:H7 and Salmonella can become internalized within growing plants and thereby are protected from any post-harvest washing treatment (Solomon et al., 2002, Watchel et al., 2002a, Cooley et al., 2003, Dong et al., 2003, Warriner et al., 2003a, Warriner et al., 2003b, Warriner et al., 2003c). To date, internalization of human pathogens in lettuce (Solomon et al., 2002, Watchel et al., 2002a), tomato plants (Guo et al., 2002), alfalfa (Gandhi et al., 2001, Dong et al., 2003), bean sprouts (Warriner et al., 2003b) and radish (Itoh et al., 1998) has been observed. However, all these studies have focused on interaction of bacteria with seedlings or sprouts exposed over short times when the potential of pathogens to become internalized is likely to be high (Warriner et al., 2003a). However, the risk posed by internalized human pathogens during extended periods of plant cultivation has yet to be established. Cooley et al. (2003) recently reported the interaction of Es. coli O157:H7 and Salmonella with growing Arabidopsis thaliana when introduced on the seed or seedling roots. The authors reported that pathogen levels increased significantly during seed germination and reached high numbers in the rhizosphere of seedlings. After prolonged cultivation in a soil-free system, Es. coli O157:H7 and Salmonella could be recovered from the entire plant, including seeds. Pathogen persistence was decreased when studies were performed in soil microcosms or by introduction of the rhizobacterium Enterobacter asburiae (Cooley et al., 2003).
In a further study, the persistence and distribution of a bioluminescent generic Es. coli strain introduced onto spinach seeds was investigated (Warriner et al., 2003c). In a gnotobiotic system, the Es. coli strain preferentially colonized the roots of emerging seedlings and became internalized. When the seedlings were cultivated hydroponically for 40 days internal Es. coli populations diminished, but those on the root and leaf surfaces persisted. When seedlings derived from non-inoculated seeds were cultivated in the presence of Es. coli, the bacterium became internalized in roots but was recovered from only the surface of leaves (Warriner et al., 2003c). Therefore, evidence to date indicates that internalization into the vascular system of plants occurs primarily in seedlings and is less prominent in mature plants. However, care has to be taken in generalizing findings as the extent of human pathogen interaction with plants is both bacterial strain and plant type specific (Dong et al., 2003). The following study investigated the interaction of virulent human pathogens with a diverse range of plant types over extended cultivation periods. The aim was to establish the microbiological risks from harvested crops that would be posed by introduction of human pathogens at the early stages of plant development.
Section snippets
Bacterial strains and inocula preparation
Bacterial strains used in this study are described in Table 1. The bioluminescent or green fluorescent markers facilitate differentiation during enumeration of pathogens. However, the bioluminescent phenotype of Es. coli O157:H7 Ph 1 was also used to visualize the in planta distribution of the pathogen.
L. monocytogenes was cultivated aerobically overnight at 37 °C in Brain Heart Infusion broth (BHI; BD Diagnostic Systems, Sparks, MD, USA) containing 5 μg/ml chloramphenicol. The other bacterial
Plasmid stability
A limitation of plasmid vectors is the ease by which they are lost from host cells in the absence of selective pressure such as that exerted by antibiotics. This is especially significant when studying bacteria/plant interaction since selective agents cannot be employed. Therefore, preliminary studies were undertaken to assess the stability of the bioluminescence or green fluorescent protein (gfp) labels. Plasmid stability within Salmonella and both strains of Es. coli O157:H7 was confirmed by
Discussion
Growth of Es. coli O157:H7 (Ph1 and C9490), S. typhimurium and L. monocytogenes on germinating seeds was observed in all the plant types tested. Growth of enteric bacteria on seedlings has been reported previously with radish (Hara-Kudo et al., 1997, Itoh et al., 1998), mung beans (Warriner et al., 2003b), alfalfa (Taormina and Beuchat, 1999, Gandhi et al., 2001), lettuce (Solomon et al., 2002, Watchel et al., 2002a) and tomato (Guo et al., 2002). The growth of human pathogens on germinating
Acknowledgements
The authors would like to thank the Ontario Ministry of Agriculture and Food for financial support for the study through their Food Safety Research Program.
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