Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/21/2008
Publication Date: 4/1/2008
Citation: Gourabathini, P., Brandl, M., Redding, K.S., Gunderson, J.H., Berk, S.G. 2008. Interactions Between Foodborne Pathogens and Protozoa Isolated from Lettuce and Spinach. Applied and Environmental Microbiology. 74:2518-2525 Interpretive Summary: Protozoa are ubiquitous in water, soil and on plants. We have investigated the interaction of ciliated protozoa with various bacterial species, with emphasis on enteric pathogens that have caused outbreaks of food-borne illness linked to leafy vegetables. Several species of protozoa have been isolated from lettuce and spinach leaves, including Glaucoma sp., Tetrahymena sp. Colpoda steinii, and amoebae. During grazing on GFP-Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes, Tetrahymena andGlaucoma, but not Colpoda, released large numbers of vesicles containing S. enterica and E. coli cells, but digested most L. monocytogenes cells. Tetrahymena produced vesicles also during grazing on Pseudomonas fluorescens and Pantoea agglomerans. The bacterial cells in the vesicles were viable as shown by Live/Dead staining and the Kogure assay. The presence of bacterivorous protozoa in the on leafy greens and their sequestration of foodborne pathogens as small aggregates in vesicles indicate that they may play an important role in the ecology of the pathogens on produce.
Technical Abstract: The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce, and to examine their interactions with S. enterica, E.coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach, and a soilborne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on GFP- or DsRed-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica, but not with L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, Colpoda steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.