Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires\' disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella\'s major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella-nematode-amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis worldwide but is rarely transferred between human hosts. Although a recognized microaerophile, the majority of C. jejuni are incapable of growing in an aerobic environment. The persistence and transmission of this pathogen outside its warm-blooded avian and mammalian hosts is poorly understood. Acanthamoebae species are predatory protists and form an important ecological niche with several bacterial species. Here, we investigate the interaction of C. jejuni 11168H and Acanthamoebae castellanii at the single-cell level. We observe that a subpopulation of C. jejuni cells can resist killing by A. castellanii, and non-digested bacteria are exocytosed into the environment where they can persist. In addition, we observe that A. castellanii can harbor C. jejuni 11168H even upon encystment. Transcriptome analyses of C. jejuni interactions revealed similar survival mechanisms when infecting both A. castellanii and warm-blooded hosts. In particular, nitrosative stress defense mechanisms and flagellum function are important as confirmed by mutational analyses of C. jejuni 11168H. This study describes a new host-pathogen interaction for C. jejuni and confirms that amoebae are transient hosts for the persistence, adaptability, and potential transmission of C. jejuni.