Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria.

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 µg/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.

Transmission electron microscopy sample preparation protocols for the ultrastructural study of cysts of free-living protozoa.

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.