Free-Living Amoebae Supporting Intracellular Growth May Produce Vesicle-Bound Respirable Doses of Legionella Within Drinking Water Systems

Abstract

Legionella pneumophila has recently been described as a major cause of water-related outbreaks in developed countries. In drinking water distribution and premise plumbing systems, L. pneumophila grow within free-living amoeba hosts in biofilm. Exposure to Legionella-containing water aerosols generated during common water usages and in built environments is responsible for causing legionellosis in humans. Here we report that the amoeba, Willaertia magna phagocytizes L. pneumophila in drinking water, supports intracellular growth, and releases L. pneumophila within vesicles of variable sizes before the trophozoites are completely lyzed to freely release the Legionella cells in the water environment. The vesicles produced in water at temperatures 22–40 °C varied in number, and ranged from 3 to 20 µm in diameter, mostly falling into the respirable size. The respirable-size vesicles that have a diameter of 3 to 10 µm may contain a maximum of 23–873 L. pneumophila cells, respectively, which could serve as a single human dose to initiate Legionella infection according to the current human dose–response information. This study suggested that current culture-based standard monitoring of drinking water for L. pneumophila would underestimate the true human health risk not only due to the inability of resolving viable but non-culturable bacterial cells but also due to underestimating the health impact of vesicle-bound Legionella cells. Thus, the regulatory guidelines for monitoring and controlling L. pneumophila should include amoebae in the surveillance of drinking water and introduce evidence-based strategies to control amoebae as an indirect and effective way of controlling legionellosis.