The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 g/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain-and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed. Foodborne pathogenic bacteria are a major cause of foodborne illness and have important implications for human public health along with economic consequences (1). Despite thorough disinfection protocols and hygiene monitoring during food production and processing, pathogenic bacteria often persist in foodrelated environments and on food, suggesting that our knowledge about the transmission routes and epidemiology of foodborne pathogenic bacteria is still incomplete.Recent studies have shown that bacteria can benefit from intracellular associations with free-living protozoa (FLP) (2, 3), heterotrophic eukaryotic microorganisms that are common in natural aquatic and terrestrial ecosystems (4). Although FLP feed on bacteria, some bacteria resist digestion. These so-called "digestion-resistant bacteria" can survive and even grow inside their FLP hosts (5). These hosts thus effectively act as a reservoir, shelter, and vector for the bacteria and can as such play an important role in their ecology (3, 6). Intracellular association with FLP has also been demonstrated for human-pathogenic bacteria (7, 8), including food-related pathogens (e.g., see references 5, 9, and 10). As FLP have been isolated from diverse food-related habitats, such as broiler houses (11-13), meat cutting plants (14), domestic refrigerators (15), and vegetables (16,17), this suggests that FLP may be implicated in the epidemiology of foodborne pathogens.Many FLP have two life cycle stages: the trophozoite and the dormant cyst. The former is the actively feeding stage, preying on bacteria, algae, viruses, yeast, and ...

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In the present study, the occurrence of free-living protozoa (FLP) and foodborne bacterial pathogens on dishcloths was investigated. Dishcloths form a potentially important source of cross-contamination with FLP and foodborne pathogens in food-related environments. First various protocols for recovering and quantifying FLP from dishcloths were assessed. The stomacher technique is recommended to recover flagellates and amoebae from dishcloths. Ciliates, however, were more efficiently recovered using centrifugation. For enumeration of freeliving protozoa on dishcloths, the Most Probable Number method is a convenient method. Enrichment was used to assess FLP diversity on dishcloths (n = 38). FLP were found on 89% of the examined dishcloths; 100% of these tested positive for amoebae, 71% for flagellates and 47% for ciliates. Diversity was dominated by amoebae: vahlkampfiids, vannellids, Acanthamoeba spp., Hyperamoeba sp. and Vermamoeba vermiformis were most common. The ciliate genus Colpoda was especially abundant on dishcloths while heterotrophic nanoflagellates mainly belonged to the genus Bodo, the glissomonads and cercomonads. The total number of FLP in used dishcloths ranged from 10 to 10 4 MPN/cm 2 . Flagellates were the most abundant group, and ciliates the least abundant. Detergent use was identified as a prime determinant of FLP concentrations on used dishcloths. Bacterial load on dishcloths was high, with a mean total of aerobic bacteria of 7.47 log 10 cfu/cm 2 . Escherichia coli was detected in 68% (26/38) of the used dishcloths, with concentrations up to 4 log 10 cfu/cm 2 . Foodborne pathogens including Staphylococcus aureus (19/38), Arcobacter butzleri (5/38) and Salmonella enterica subsp. enterica ser. Halle (1/38) were also present. This study showed for the first time that FLP, including some opportunistic pathogens, are a common and diverse group on dishcloths. Moreover, important foodborne pathogens are also regularly recovered. This simultaneous occurrence makes dishcloths a potential risk factor for cross-contamination and a microbial niche for bacteria-FLP interactions.

Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.

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