Protozoa are an integral part of most microbial consortia and are ubiquitous in nature, particularly in environments where water is present. Many species of protozoa feed on bacteria, which they ingest by phagocytosis and sequester within food vacuoles. Bacterivorous protozoa have been termed the "Trojan horses" of the microbial world as they may shield pathogenic bacteria that they internalize from the early defense responses of human hosts (2). Recent reviews report over 23 species and groups of pathogens found to associate with amoebae (16,27). Passage of intracellular pathogens such as Legionella pneumophila and Mycobacterium avium through Acanthamoeba castellanii results in these pathogens' greater infectivity (11, 12). Barker et al. The role of protozoa in the virulence of food-borne pathogens and their persistence in the environment has been relatively unexplored. Several pathogenic coliform species were found to be more resistant to chlorination while internalized within A. castellanii and Tetrahymena pyriformis (17). In addition, multiplication of Escherichia coli O157:H7 within Acanthamoeba polyphaga has been reported (4). However, the significance of bacterial entrapment within protozoa cysts or vesicles in the contamination cycle of food-borne pathogenic bacteria has not been addressed. The objective of our study was to determine whether Salmonella enterica could be released in a viable form in vesicles of Tetrahymena and whether this internalization affords a survival advantage over those bacteria that remain free in an aquatic environment. MATERIALS AND METHODSStrains and culture. S. enterica serovar Thompson strain RM1989 is a clinical isolate associated with an outbreak linked to cilantro in California (6, 9). Serovar Thompson strain STN pGT-Kan is a spontaneous nalidixic-acid-resistant mutant of strain RM1989 that is intrinsically labeled with the green fluorescent protein (GFP) by transformation with plasmid pGT-Kan. Plasmid pGT-Kan was constructed by fusion of the nptII promoter fragment from Tn5 to gfp on pPROBE-GT (22) and is stably maintained in serovar Thompson. Strain STN pGT-Kan was used throughout this study and cultured with agitation at 28°C in Luria-Bertani broth (without NaCl) containing gentamicin at 15 g ml Ϫ1 . Listeria monocytogenes strain RM2194 pNF8 is a clinical isolate labeled with GFP and was described previously (15). L. monocytogenes pNF8 was cultured at 28°C in Luria-Bertani broth (without NaCl) containing erythromycin and lincomycin at 1 and 25 g ml Ϫ1 , respectively. Tetrahymena was isolated from soil in California, and the isolate was named SSU. Enrichment for Tetrahymena SSU was performed by the addition of E. coli strain DH5-␣ cells to aqueous soil suspensions. An axenic culture of this isolate was then obtained by incubation of the suspension in Neff's culture medium supplemented with penicillin G and streptomycin sulfate at 250 g ml Ϫ1 each

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, Escherichia 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 soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent proteinor red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the 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 during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. 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.

Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophilacells. Vesicles ranged from 2.1 to 6.4 μm in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.

The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of the gram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in free spherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either within intracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as a function of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulsechase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feeding ciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of the ingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence system were efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pellets produced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls. The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they were outer membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutants produced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system to resist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila depends on bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication.

The intracellular bacterial pathogen Legionella pneumophila follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that L. pneumophila survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate Tetrahymena tropicalis before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through T. tropicalis, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene rpoS, or the response regulator gene letA, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through T. tropicalis. Therefore, T. tropicalis has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The Tetrahymena model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.