Enhanced Survival of Salmonella enterica in Vesicles Released by a Soilborne Tetrahymena Species

Abstract

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

Salmonella transcriptional signature in Tetrahymena phagosomes and role of acid tolerance in passage through the protist

“…We have reported previously that S. Typhimurium can survive digestion by Tetrahymena at high rates and is released as viable cells in its fecal pellets in which it has enhanced survival compared with cells remaining undigested and free in suspension (Brandl et al, 2005). This is in contrast with L. monocytogenes, which is digested by the protist and detected infrequently in its fecal pellets (Brandl et al, 2005;Gourabathini et al, 2008).…”

“…This is in contrast with L. monocytogenes, which is digested by the protist and detected infrequently in its fecal pellets (Brandl et al, 2005;Gourabathini et al, 2008). In addition, S. Typhimurium does not decrease the viability of Tetrahymena during its intravacuolar passage (Gourabathini et al, 2008).…”

“…Tetrahymena sp. MB125 was isolated from soil in California, belongs to an unknown species of this genus and has been described previously (Brandl et al, 2005). It was used in this study because of its release of numerous large fecal pellets containing viable S. enterica cells upon grazing on this pathogen (Figure 1).…”

“…After co-incubation of SL1344 and Tetrahymena MB125 as described above, the suspension was filtered through Millipore black Isopore membranes (0.2 mm pore size, 25 mm diameter) and cell viability assessed as described previously (Brandl et al, 2005). Briefly, filters were gently submerged in H 2 O to allow Tetrahymena cells to swim away from the filter leaving behind free bacteria and pellets.…”

Co-occurrence of free-living protozoa and foodborne pathogens on dishcloths: Implications for food safety

“…However intraprotozoan survival and/or replication depend on various factors such as bacterial strain and environmental conditions (Schuppler, 2014;Vaerewijck et al, 2014). As a result, FLP can act as vectors, introducing pathogens into novel habitats, or as transmission routes toward hosts (Berk et al, 1998;Bouyer et al, 2007;Brandl et al, 2005;Matz and Kjelleberg, 2005;Snelling et al, 2008). In addition, they can also act as a protective niche or shelter for bacteria against harsh environmental conditions (Barker and Brown, 1994;King et al, 1988;Snelling et al, 2005), or even as training grounds and evolutionary cribs for foodborne pathogens (Molmeret et al, 2005), enhancing bacterial virulence and mediating bacterial gene transfer.…”