Optimized methods for Legionella pneumophila release from its Acanthamoeba hosts

Background: Free-living amoebae (FLA) and particularly acanthamoebae serve as vehicles and hosts for Legionella pneumophila, among other pathogenic microorganisms. Within the amoebae, L. pneumophila activates a complex regulatory pathway that enables the bacteria to resist amoebal digestion and to replicate. Moreover, the amoebae provide the bacteria protection against harsh environmental conditions and disinfectants commonly used in engineered water systems. To study this ecological relationship, co-culture and infection models have been used. However, there is a lack of data regarding the effectiveness of the different methods used to release intracellular bacteria from their amoebal hosts. The aim of this study was to evaluate the impact of the methods used to release intracellular L. pneumophila cells on the culturability of the bacteria. Furthermore, the standard method ISO 11731:1998 for the recovery and enumeration of Legionella from water samples was evaluated for its suitability to quantify intracellular bacteria. Results: The effectiveness of the eight release treatments applied to L. pneumophila and Acanthamoeba strains in a free-living state varied between bacterial strains. Moreover, the current study provides numerical data on the state of co-culture suspensions at different time points. The release treatments enhanced survival of both microorganisms in co-cultures of L. pneumophila and Acanthamoeba. Passage through a needle (21G, 27G) and centrifugation at 10,000 x g showed the highest bacterial counts when releasing the bacteria from the intracellular state. Regarding the ISO 11731:1998 method, one of the tested strains showed no differences between the recovery rates of associated and free-living L. pneumophila. However, a reduced bacterial recovery rate was observed for the second L. pneumophila strain used, and this difference is likely linked to the survival of the amoebae. Conclusions: Mechanical release treatments were the most effective methods for providing bacterial release without the use of chemicals that could compromise further study of the intracellular bacteria. The current results demonstrated that the recovery of L. pneumophila from water systems may be underestimated if protozoal membranes are not disrupted.