Abstract
BackgroundFree-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.ResultsThe 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 × 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.ConclusionsMechanical 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.
Highlights
Free-living amoebae (FLA) and acanthamoebae serve as vehicles and hosts for Legionella pneumophila, among other pathogenic microorganisms
Free-living amoebae (FLA), acanthamoebae, have recently gained scientific attention because of their intrinsic pathogenicity [1,2,3] and because they serve as vehicles and hosts for a wide range of pathogenic microorganisms, such as Coxiella burnetti, Chlamydophila pneumoniae, Mycobacterium spp., Pseudomonas aeruginosa, Vibrio cholera and Legionella pneumophila [4,5,6,7]
Effect of release treatments on pure cultures To evaluate potential treatments for the release of intracellular bacteria, suspensions of two different L. pneumophila strains and two different Acanthamoeba strains were initially tested in a free-living state
Summary
Free-living amoebae (FLA) and acanthamoebae serve as vehicles and hosts for Legionella pneumophila, among other pathogenic microorganisms. 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. Free-living amoebae (FLA), acanthamoebae, have recently gained scientific attention because of their intrinsic pathogenicity [1,2,3] and because they serve as vehicles and hosts for a wide range of pathogenic microorganisms, such as Coxiella burnetti, Chlamydophila pneumoniae, Mycobacterium spp., Pseudomonas aeruginosa, Vibrio cholera and Legionella pneumophila [4,5,6,7] Among these bacteria, the relationship between Acanthamoeba and L. pneumophila is one of the most studied due to the associated health risk. There is a lack of data regarding the different treatments and their effects on L. pneumophila
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