Relationship between free amoeba and Legionella: studies in vitro and in vivo.

In 1980, Robowtham demonstrated that Legionella multiplies in free amoeba cytoplasm and hypothesized that the amoeba could act as a reservoir of virulent bacteria. In this paper we report various aspects of the relationship between amoeba and Legionella. A liquid medium co-culture method was applied to Acanthamoeba sp. and Legionella pneumophila serogroup 1. Within 4 days, Legionella growth increased by 2 log s CFU/ml. Using a direct immunofluorescence assay and electron microscopy, Legionella was shown to grow abundantly inside phagosomes, and bacteria and/or antigen were present on the cytoplasmic membrane of the amoeba. These aspects are very similar to those observed with Legionella-infected alveolar macrophages. The morphology and structure of Legionella cells were modified after 20 days of co-culture: - viable bacteria showed large fatty cytoplasmic inclusions, - gas liquid chromatography analysis demonstrated a decrease in the i16:0 fatty acid ratio. Cystic forms of amoeba were abundant but none contained viable Legionella. In an in-vivo study using a guinea-pig aerosol infection model, we compared the virulence of Legionella in co-culture with Legionella grown on charcoal dialysed yeast extract (CDYE) agar medium. The Legionella obtained by co-culture had an LD 50 (50% lethal dose) similar to that obtained for those grown on CDYE, showing that bacterial virulence is preserved in the cellular model.